A di‐leucine‐based motif in the cytoplasmic tail of LIMP‐II and tyrosinase mediates selective binding of AP‐3

Stefan Höning; Ignacio V. Sandoval; Kurt von Figura

doi:10.1093/emboj/17.5.1304

A di‐leucine‐based motif in the cytoplasmic tail of LIMP‐II and tyrosinase mediates selective binding of AP‐3

Höning, Stefan; Sandoval, Ignacio V.; von Figura, Kurt 1998-03-02 00:00:00 The EMBO Journal Vol.17 No.5 pp.1304–1314, 1998 A di-leucine-based motif in the cytoplasmic tail of LIMP-II and tyrosinase mediates selective binding of AP-3 1 2 membrane proteins and its function is not yet understood Stefan Ho¨ning , Ignacio V.Sandoval and (Barrinocanal et al., 1986; Vega et al., 1991). Unlike the Kurt von Figura proteins of the lamp family, which contain a tyrosine- Institute for Biochemistry II, University of Go¨ttingen, Gosslerstr. 12d, based sorting signal of the GYXXZ type (Z being either 37073 Go¨ttingen, Germany and Centro de Biologia Molecular I, F, L or V) mediating intracellular sorting (Ho¨ning ‘Severo Ochoa’, Universidad Autonoma de Madrid, Madrid, Spain and Hunziker, 1995; Ho¨ning et al., 1996; Gough and Corresponding author Fambrough, 1997), the LIMP-II cytoplasmic tail harbors an LI signal of the di-leucine type. This signal has been Among the various coats involved in vesicular trans- shown to be necessary for the intracellular targetting of port, the clathrin associated coats that contain the the protein to lysosomes (Vega et al., 1991; Sandoval adaptor complexes AP-1 and AP-2 are the most et al., 1994). Though LIMP-II has been shown to be extensively characterized. The function of the recently packed into an as yet undeﬁned type of coated vesicles at described adaptor complex AP-3, which is similar to the TGN (Barriocanal et al., 1986), further details of the AP-1 and AP-2 in protein composition but does not intracellular pathways of LIMP-II are not known. associate with clathrin, is not known. By monitoring Clathrin-coated vesicles (CCVs) were the ﬁrst type surface plasmon resonance we observed that AP-3 is of intracellular vesicles to be identiﬁed and characterized. able to interact with the tail of the lysosomal integral Clathrin is the major structural component of CCVs; membrane protein LIMP-II and that this binding the speciﬁcity of its function is mediated by the depends on a DEXXXLI sequence in the LIMP-II tail. associated adaptor complexes AP-1 and AP-2 (reviewed Furthermore, AP-3 bound to the cytoplasmic tail of in Pearse and Robinson, 1990). Coated pits and coated the melanosome-associated protein tyrosinase which vesicles that are formed during endocytosis only contains a related EEXXXLL sequence. The tails of contain the AP-2 complex, while the same type of LIMP-II and tyrosinase either did not interact, or only coated pits and coated vesicles that derive from the interacted poorly, with AP-1 or AP-2. In contrast, the TGN contain the AP-1 complex (Robinson, 1987). The cytoplasmic tails of other membrane proteins con- four subunits that build up the AP-1 complex are the taining di-leucine and/or tyrosine-based sorting signals large γ- and β1-subunits together with the μ1-medium did not bind AP-3, but AP-1 and/or AP-2. This points and the σ1-small subunit. The related AP-2 complex to a function of AP-3 in intracellular sorting to lyso- consists of the α- and β2-subunits that assemble together somes and melanosomes of a subset of cargo proteins with the μ2- and σ2-subunits (Ahle et al., 1988; via di-leucine-based sorting motifs. Schro¨der and Ungewickell, 1991; Traub, 1997). The Keywords: biosensor/coated vesicles/endosome/ two complexes display signiﬁcant homology to each membrane trafﬁc/protein sorting other and in addition exhibit the similar structure of a large trunk that is separated from appendices (‘ears’) by a ﬂexible hinge. A major function of both adaptor complexes is to promote clathrin-lattice formation onto Introduction the respective membrane (Wilde and Brodsky, 1996). This function seems likely to involve the large subunits The lysosomal integral membrane protein LIMP-II γ- and β1 of AP-1 and α- and β2 of the AP-2 complex. (Barrinocanal et al., 1986; Vega et al., 1991), originally A second important function of AP-1 and AP-2 is the isolated from rat cells, belongs to a group of proteins that interaction with sorting signals that are present in the is highly enriched in lysosomal membranes. This group cytoplasmic tails of cargo proteins to be concentrated also comprises membrane proteins of the lamp-1, lamp-2 in CCVs (for review see Kirchhausen et al., 1997; and lamp-3 family, and lysosomal acid phosphatase (LAP) Robinson, 1997). Studies using different experimental (reviewed in Hunziker and Geuze, 1996). LIMP-II is a systems have revealed that tyrosine-based sorting signals type-III protein that traverses the membrane twice, with of membrane proteins such as the LDL receptor (Pearse, an N-terminal transmembrane anchor as a result of an 1988), LAP (Sosa et al., 1993), mutant hemagglutinin uncleaved signal peptide and a second membrane spanning (Heilker et al., 1996) and lamp-1 (Ho¨ning et al., 1996), region near the C-terminus, followed by a 20 amino acid as well as di-leucine-based sorting signals of membrane cytoplasmic tail. Most of the protein loops into the proteins such as the MPR300 (Glickman et al., 1989), lysosome where ﬁve cysteine residues may be involved mutant hemagglutinin (Heilker et al., 1996), invariant in disulﬁde bond formation. This region also contains 11 potential N-glycosylation sites. Glycosylation of the chain (Salamero et al., 1996) and MPR46 (Mauxion protein results in an apparent molecular weight of 74 kDa et al., 1996; Honing et al., 1997), can be recognized for the 477 amino acid mature protein. LIMP-II does by adaptor complexes. The situation could be more not display homology to any of the other lysosomal complex for proteins that contain more than one sorting 1304 © Oxford University Press LIMP-II tail and tyrosinase tail binding to AP3 signal in their tail, such as the LDL receptor (Matter Results et al., 1992, 1994) and the MPRs (Denzer et al., 1997; The LIMP-II tail is not recognized by AP-1 and AP-2 Ho¨ning et al., 1997), and it is not clear whether It has recently been shown that the lysosomal membrane adaptors recognize only a single motif or if they protein lamp-1 binds through a tyrosine-based sorting simultaneously interact with multiple sequence motifs. signal in its cytoplasmic tail to AP-1 and AP-2 with With the yeast two-hybrid system it was possible to high afﬁnity (Ho¨ning et al., 1996). In addition, electron show that different types of tyrosine-based sorting microscopic analysis of cells expressing wild-type (wt) or signals can be recognized by the μ-chains of AP-1 and mutant lamp-1 have shown a co-localization of lamp-1 AP-2 (Ohno et al., 1995, 1996; Boll et al., 1996). and AP-1, and the appearance of lamp-1 in TGN-derived However, it is still not clear if membrane proteins clathrin-coated vesicles. LIMP-II shares with lamp-1 the containing di-leucine-based sorting signals also interact direct routing to lysosomes, bypassing the cell surface with the μ-chain, or if one of the other subunits is (Vega et al., 1991; Sandoval et al., 1994). Unlike involved in the recognition of this type of sorting signal. lamp-1, the cytoplasmic tail of LIMP-II lacks a tyrosine- Apart from AP-1 and AP-2, which promote the forma- based sorting signal. Targetting of LIMP-II to lysosomes tion of clathrin-coated pits and vesicles at the plasma depends on a pair of LI residues which belongs to the membrane and at the TGN, many organelles do not seem class of di-leucine-based sorting motifs (Sandoval and to contain a clathrin coat yet vesicular transport to and Bakke, 1994; Sandoval et al., 1994). from all organelles is a prerequisite for the viability of To analyze a possible interaction of the LIMP-II cyto- plasmic tail with puriﬁed AP-1 and AP-2, we utilized a the living cell. This has led to the hypothesis that other biosensor system monitoring surface plasmon resonance protein coats beside clathrin may exist that have a similar (SPR). This method has been used in several studies to function in membrane trafﬁc (Stoorvogel et al., 1996; analyze the interaction between puriﬁed adaptors and the Robinson, 1997). The COP-I and COP-II coats that are EGF-receptor (Boll et al., 1995), the hemagglutinin tail involved in transport between the ER and the Golgi are (Heilker et al., 1996) and the lamp-1 tail (Ho¨ning et al., two examples of recently characterized non-clathrin coats 1996), and to deﬁne the AP-1 and AP-2 binding sites (for review see Cosson and Letourneur, 1997). The identi- on the MPR46 tail (Ho¨ning et al., 1997). A peptide ﬁcation of novel coats that may have a function in corresponding to the full-length LIMP-II tail (Figure 1) membrane protein sorting along their intracellular trafﬁc was synthesized, together with a mutant tail peptide where routes is a subject of active research (Stoorvogel et al., the leucine of the LI-sorting signal of LIMP-II is replaced 1996; Robinson, 1997). by glycine (LIMP-II L18G; Figure 1). This substitution Based on a signiﬁcant sequence homology to the known was previously shown to disrupt the lysosomal sorting of AP-1/AP-2 subunits and the search of EST databases, four LIMP-II (Sandoval et al., 1994). As a positive control we subunits of a novel adaptor-like complex named AP-3 used a peptide corresponding to the tail of MPR46, which have recently been cloned and characterized (Newman is known to bind to AP-1 and AP-2 (Sosa et al., 1993; et al., 1995; Dell’Angelica et al., 1997a; Simpson et al., Honing et al., 1997). The LIMP-II peptides were synthe- 1997). Like AP-1 and AP-2, AP-3 is a protein hetero- sized with an additional N-terminal cysteine residue which tetramer, which is composed of δ-adaptin (160 kDa), allows coupling of the peptides to the sensor chip surface β3-adaptin (140 kDa), the medium chain μ3 (47 kDa) and via this residue (see Materials and methods). All peptides the small chain σ3 (22 kDa). The complex is ubiquitously were coupled to a CM-5 sensor chip in equal densities expressed, with special variants of β3(β-NAP) and μ3 2 (~0.2 pmol/mm per peptide, data not shown) and tested (p47B) being expressed in brain. In contrast to AP-1 and for their ability to bind to puriﬁed AP-1 and AP-2. As AP-2, the AP-3 complex does not associate with clathrin. shown in Figure 2, only the MPR46 tail interacted with Immunoﬂuorescence analysis has revealed a distribution AP-1 (K 13 nM) and AP-2 (K 17 nM). In contrast, D D of AP-3 in the Golgi region and in peripheral structures neither the wt LIMP-II peptide nor the LIMP-II L18G that are thought to represent endosomes (Newman et al., peptide interacted detectably with AP-1 or AP-2 at 1995; Simpson et al., 1997). By the use of the yeast two- adaptor concentrations of up to 500 nM (shown for 100 nM hybrid system it was demonstrated that μ3 can interact AP-1 or AP-2, Figure 2). with tyrosine-based sorting signals similar to the clathrin- associated counterparts μ1 and μ2 in AP-1 and AP-2 Brain cytosol contains a component with LIMP-II (Dell’Angelica et al., 1997b). tail binding activity In an attempt to characterize the adaptor complexes Since we did not detect any binding of AP-1 or AP-2 to that bind to the cytoplasmic tail of LIMP-II, we observed the LIMP-II tail, we examined whether or not other that LIMP-II interacts with AP-3 but not with AP-1 or cytosolic components interact with the LIMP-II cyto- AP-2. This interaction is speciﬁc and dependent on the plasmic tail in a manner that is dependent on the LI LI motif that is critical for sorting of LIMP-II. In addition, sorting signal. For this purpose, cytosol from pig brain the acidic residues DE in positions –4 and –5 to the LI was prepared. When the brain cytosol was passed over signal appear to modulate this interaction. Furthermore, the MPR46 tail-derived sensor surface, a strong interaction the cytoplasmic tail of tyrosinase, a membrane protein of was observed suggesting the presence of functional lysosome-related melanosomes which contains a similar adaptors (see below and Figure 5). A similar interaction di-leucine motif, was also found to interact with AP-3. was monitored for the wt LIMP-II tail, while only a We propose a role of AP-3 in the sorting of a subset of reduced interaction was detected for the mutant LIMP- lysosomal and melanosomal membrane proteins. II L18G tail peptide (Figure 3). In the presence of 1305 S.Ho¨ning, I.V.Sandoval and K.von Figura Fig. 1. Amino acid sequences of cytoplasmic tail peptides. Peptides corresponding to the cytoplasmic tails of wt and mutant LIMP-II, lamp-1, LAP, tyrosinase and MPR46 are shown in the one letter code with the C-terminal end to the right. The open boxes on the left represent the junction of the membrane spanning region and the cytoplasmic tail. Residues known to be critical for sorting, and the corresponding mutations, are indicated by bold letters. All peptides represent the full-length cytoplasmic tails of the respective proteins except that of tyrosinase. The tyrosinase peptide lacks the membrane proximal residues 1–5. All peptides were immobilized on the sensor chip surface involving primary amino or thiol groups (see Materials and methods). carboxymethylated dextran, which minimizes unspeciﬁc binding to the chip surface which itself contains carboxy- methylated dextran, the residual binding to the mutant LIMP-II tail was negligible (Figure 3). In contrast, the signal obtained with the wt LIMP-II tail and the MPR46 tail peptide was only slightly reduced indicating the speciﬁcity of the interaction. Thus pig brain cytosol contains one or more components that have the ability to bind to the LIMP-II tail in a manner that is dependent on the LI sorting signal. Fig. 2. Binding of puriﬁed AP-1 and AP-2 to LIMP-II and MPR46 tail peptides. AP-1 and AP-2 preparations were passed over MPR46-, wt AP-3, but not AP-1 or AP-2, binds to the LIMP-II LIMP-II- and LIMP-II L18G-derived chip surfaces for 2 min tail (association) before the chip surface was washed with buffer A for The experiments described above demonstrated that a 2 min (dissociation). Only MPR46 was able to interact with both AP-1 and AP-2, whereas wt and mutant LIMP-II did not interact with either cytosolic factor(s) exists which can bind speciﬁcally to adaptor complex. the LIMP-II tail. To further analyze the cytosolic component(s) that binds to the cytoplasmic tails of MPR46 and LIMP-II, we fractionated pig brain cytosol LIMP-II from that binding to MPR46. Fractions 23–26, by gel ﬁltration. All fractions obtained were then while exhibiting an interaction with the MPR46 tail passed over sensor surfaces derived with the MPR46 peptide, did not interact at all with the LIMP-II peptides. tail, with wt LIMP-II or the LIMP-II L18G mutant tail On the other hand, fractions 28–30 exhibited binding peptide. To compare the binding, the resonance units activity for the LIMP-II tail but not for the MPR46 that remained bound to the chip-surface after a 2 min tail. Furthermore, the L18G mutant peptide was only association/2 min dissociation cycle were plotted. It poorly recognized by fractions 28–30. No other fractions should be noted that this experiment was performed besides those plotted in Figure 4 were found to bind without quenching non-speciﬁc interaction by adding to the tail peptides. carboxymethylated dextran. As shown in Figure 4, we As it had already been shown that MPR46 binds to observed a separation of the components that bind to both clathrin adaptors, it was suspected that fractions 1306 LIMP-II tail and tyrosinase tail binding to AP3 consistent with the ﬁnding that the LIMP-II tail does not interact with puriﬁed AP-1 or AP-2 (Figure 2). To further corroborate these ﬁndings, crude brain cytosol was passed over MPR46 and LIMP-II-derived surfaces for a 2 min association cycle. After a 2 min buffer wash (dissociation), antibodies that recognize native AP-2 or AP-3 were passed over the chip surface. As shown in Figure 5, when cytosol had been passed over the MPR46 tail-derived surface a strong signal was obtained with the anti-AP-2 antibody, indicating that AP-2 is recruited to the tail. In contrast, no binding of the anti-AP-3 antibody was observed, in agreement with the observation that AP-3 does not bind to MPR46. On the other hand, when crude brain cytosol was applied to the LIMP-II-derived surface a speciﬁc interaction with anti-AP-3 was observed, but not with anti-AP-2 (or anti- AP-1, data not shown). Thus the perfusion of adaptor- speciﬁc antibodies over the tail-derived chip surfaces that were ﬁrst allowed to recruit the adaptors from cytosol conﬁrmed that AP-3 binds to the LIMP-II tail. AP-3 binding to LIMP-II requires an acidic cluster in addition to the LI sorting motif It has previously been shown in COS cells transfected with tail-mutants of LIMP-II that the LI motif is critical for correct intracellular sorting. In addition, mutation of the aspartic and glutamic acidic residues at positions –5 and –4 relative to the LI motif also interferes with correct Fig. 3. Binding of pig brain cytosol to the LIMP-II cytosplasmic tail. Crude brain cytosol from pig brain was prepared and passed in the lysosomal targetting of LIMP-II (Sandoval et al., 1994; presence or absence of 1 mg/ml carboxymethylated dextran over tail- S.Martinez-Arca and I.V.Sandoval, submitted). In order to peptide derived sensor chip surfaces to record interaction. MPR46 and deﬁne further the sequence determinants in the LIMP-II wt LIMP-II were able to bind a component(s) of brain cytosol, tail that mediate AP-3 binding, a set of mutant tail peptides whereas binding to mutant LIMP-II L18G was of low afﬁnity. Carboxymethylated dextran, which reduces non-speciﬁc binding to the of LIMP-II (Figure 1) was synthesized to analyze their chip surface, abolished almost completely the residual binding to binding to AP-3 by SPR (see above and Figure 6). The LIMP-II L18G. importance of leucine 475 for AP-3 binding has already been shown since the control peptide of the LIMP-II tail, in which the leucine is replaced by glycine (LIMP-II 23–26 may contain AP-1 and AP-2. All fractions obtained L18G), does not bind AP-3 (Figures 3 and 4). Furthermore, from gel ﬁltration were subjected to SDS–PAGE and when a truncated tail-peptide of LIMP-II lacking the C- Western blotting, followed by incubation with anti- terminal pentapeptide PLIRT was immobilized, binding bodies speciﬁc for the known adaptor complexes. To of AP-3 was totally abolished (data not shown). This detect the AP-1 complex, a monoclonal antibody against result is in agreement with the data obtained from cells the γ-subunit was applied; the AP-2 complex was detected expressing the corresponding truncated LIMP-II protein by an α-subunit speciﬁc antibody; and the AP-3 complex which is delivered to the cell surface (Sandoval et al., was identiﬁed by antiserum speciﬁc for the δ-subunit 1994). (Simpson et al., 1997). As shown in Figure 4, the When the two amino acid residues DE, in positions AP-2 speciﬁc antibody only reacted with fractions 23–26. –4 and –5 relative to the LI motif, were mutated An identical pattern was observed with an antibody separately to alanines, a 20- to 60-fold decrease in against AP-1 (data not shown). In contrast, the anti-AP-3 afﬁnity to AP-3 as compared with the wt LIMP-II tail antibody reacted with fractions 28–30, but not with frac- was observed (Figure 6A). Moreover, the replacement tions 23–26. Thus, gel ﬁltration of brain cytosol leads to of both residues by arginine led to a 100-fold decrease the separation of AP-1/AP-2 from AP-3. The apparent in binding afﬁnity to AP-3. These results indicate that size of AP-3 as compared with a molecular weight both of the two acidic residues preceding the LI motif standard matched a molecular weight of ~350 kDa. In in the LIMP-II tail at positions –4 and –5 are necessary contrast, the clathrin-associated adaptor complexes for a high afﬁnity interaction with the AP-3 complex. appeared to run as a complex slightly larger than expected Another explanation would be that mutations within the from their calculated weight, which is comparable with tail peptide disrupt the structure of the immobilized that of AP-3. This unusual behaviour during gel ﬁltration peptide, thereby causing loss of adaptor binding. of AP-1 and AP-2 has been noted previously (Keen To assess the plausibility of this assumption, three et al., 1979; Keen 1987). Most interestingly, the LIMP-II different mutant LIMP-II tail peptides were synthesized tail is only recognized by factors present in the AP-3 (LIMP-II S6A, E9Q and T11A) which correspond to enriched fractions but does not show any interaction with LIMP-II mutants that are known to be correctly sorted AP-1/AP-2-containing fractions (Figure 4). This result is in vivo (Sandoval et al., 1994). As shown in Figure 1307 S.Ho¨ning, I.V.Sandoval and K.von Figura Fig. 4. Gel ﬁltration analysis of pig brain cytosol. Cytosol from pig brain was fractionated by gel ﬁltration on Superdex-200. The fractions were analyzed by SDS–PAGE and Western blotting, and probed with antibodies to the α-subunit of AP-2 and the δ-subunit of AP-3 as indicated in the ﬁgure. Furthermore, each fraction was tested for interaction with the MPR46, wt LIMP-II and LIMP-II L18G-derived sensor chip surfaces. The bars represent the resonance units that remained associated to the chip after an association (2 min)/dissociation (2 min) cycle. No other fraction, apart from those indicated, exhibited any interaction with the tail peptides. The values presented are corrected for background binding which was below 55 RU in all fractions. 6B, the observed binding of AP-3 to these mutant tail peptides was nearly identical to that observed for wt LIMP-II, indicating that mutations within the tail peptide do not lead a priori to loss of adaptor binding. In addition, previous NMR analysis of the LIMP-II tail peptide (Sandoval et al., 1994) has revealed the predominance of random coil conformations indicating a high ﬂexibility of the LIMP-II tail. Taken together, the data obtained with the biosensor conﬁrm the in vivo data showing that sorting of LIMP-II is dependent on a di-leucine motif which functions in the context of two acidic residues. The observation that substitutions of several residues known not to interfere with sorting of LIMP-II also do not affect the interaction of the LIMP-II tail with AP-3 underlines the signiﬁcance of the in vitro data. The speciﬁcity of the AP-3 LIMP-II tail interaction was Fig. 5. AP-3 is the cytosolic component that binds to the LIMP-II tail peptide. A cytosol fraction enriched in AP-3 was passed over a LIMP- also conﬁrmed by the perfusion of the LIMP-II-derived II-derived chip surface (left) and an AP-2 enriched cytosol fraction chip with an anti-LIMP-II tail antiserum prior to the over a MPR46-derived chip surface (right). After an association/ injection of the AP-3 enriched fraction. The antiserum, dissociation cycle, antibodies speciﬁc for AP-3 (δ-subunit) or AP-2 which speciﬁcally recognizes the C-terminal 6 amino acids (α-subunit) were injected. Note that LIMP-II is recruiting AP-3 but not AP-2 from the cytosol, as revealed by the positive signal obtained of the LIMP-II tail, was injected at a low ﬂow-rate to by the anti-AP-3 antibody. On the other hand, the MPR46 recruits obtain maximal binding. Subsequently, an AP-3-enriched AP-2 but not AP-3 from the cytosol, as indicated by the strong signal fraction (fraction 29, see Figure 4) was passed over the with the anti-AP-2 antibody. surface. The antibody perfusion led to a 70% loss in binding of AP-3, while perfusion with a control serum did not interfere with the subsequent binding of AP-3 to the LIMP-II tail (Figure 7). It should be noted that the tail-speciﬁc antiserum to a hexapeptide comprising the LIMP-II tail antibody does not cross-react with the mutant LI-sorting motif leads to the inhibition of AP-3 interaction. tail-peptide L18G (data not shown). Thus binding of the Taken together the experiments described above demon- 1308 LIMP-II tail and tyrosinase tail binding to AP3 Fig. 7. Binding of AP-3 to the LIMP-II tail is sensitive to the injection of anti-LIMP-II tail antiserum. An anti-LIMP-II tail antiserum or a control serum was passed over the LIMP-II-derived chip. To obtain maximum binding of the antiserum to the tail-peptide the ﬂow-rate was reduced to 5 μl/min. After antibody washing was extended to 6 min to reach a near constant resonance, an AP-3 enriched cytosol fraction (black curve, see Figure 4) was passed over the chip surface, followed by a wash with buffer. As a control, the dissociation of the Fig. 6. Acidic residues preceding the LI motif in the LIMP-II tail are antibodies from the LIMP-II tail peptide was recorded separately for necessary for high afﬁnity binding of AP-3. LIMP-II tail peptides with up to 10 min (grey curve). Note that AP-3 binding following mutations of the acidic residues D12 and/or E13 (Figure 1) were 2 saturation of the chip surface with the anti-LIMP-II tail antiserum (A) coupled to the CM-5 sensor chip in equal densities (0.2 pmol/mm was reduced by 70% of the control (B). 10%). The derived surfaces were then tested for their ability to interact with an AP-3 enriched cytosol fraction (A). Tail peptides that correspond to LIMP-II mutants known not to interfere with sorting in vivo served as controls (B). The kinetic values for the on-rate (k ), bearing Staphylococcus aureus cells. Control cytosol was the off-rate (k ) and the equilibrium constant K (k /k ) represent d D a d treated likewise except that the anti-AP-3 antiserum was relative afﬁnities to that of the wt LIMP-II tail set to 1 (C). omitted (see Materials and methods). Identical aliquots of immuno-depleted and control cytosol were then analyzed for binding to the wt LIMP-II as well as to the mutant strate that a DEXXXLI-sorting motif in the LIMP-II tail LIMP-II tail, and to the MPR46 tail. As shown in Figure mediates a high afﬁnity interaction with AP-3. 8, after the incubation without antibody the cytosol retained 80% of its original binding activity as revealed by incuba- AP-3 is the major cytosolic factor that has a tion with the LIMP-II and the MPR46 tail peptides. LIMP-II binding capacity However, if the cytosol was depleted from AP-3 by The experiments described above provide evidence that incubation with the speciﬁc antibody prior to SPR analysis, the AP-3 complex becomes bound to the cytoplasmic tail binding of the cytosol to the wt LIMP-II tail was reduced of LIMP-II. However, it is possible that additional cytosolic by 75%. The residual binding is in the same range as factors contribute to the binding activity detected by SPR that observed for the binding of immuno-depleted or and that binding of AP-3 may even be of an indirect control cytosol to the L18G mutant tail-peptide. It nature. To test this possibility, cytosol was immuno- should be noted that immuno-depleted cytosol retains its depleted of AP-3. If cytosolic factors apart from AP-3 ability to bind to the MPR46 tail, indicating the speciﬁc bind to the LIMP-II tail, immuno-depletion of AP-3 from depletion of AP-3. These results indicate that AP-3 cytsosol should only partially reduce the binding activity. The brain cytosol was depleted from AP-3 by two rounds accounts for essentially all of the speciﬁc binding activity of incubation with anti-AP-3 antiserum and protein A, to the LIMP-II tail as detected by SPR. 1309 S.Ho¨ning, I.V.Sandoval and K.von Figura Fig. 8. Cytosol depleted from AP-3 loses the LIMP-II tail binding activity. Crude brain cytosol was precleared by incubation for 1 h with S.aureus followed by centrifugation. The supernatant was divided into three aliquots. One was used directly for SPR analysis (open bars). The other aliquots were subjected to two cycles of immuno-depletion with anti-AP-3 antiserum pre-adsorbed to S.aureus (ﬁlled bars) or Fig. 9. Tail peptides that can interact with AP-3 in vitro. The wt and S.aureus alone (hatched bars) prior to SPR analysis (see Materials and mutant tail peptides of tyrosinase (A and B), LAP and lamp-1 (C and methods). Binding activity of pre-cleared cytosol to the LIMP-II tail or D) were immobilized on the sensor chip and analyzed for their binding to the MPR46 tail (determined as in Figure 4) was set to 100% and capacity of AP-1/AP-2 (A and C) and AP-3 (B and D) enriched used as reference for the other binding activities. Immuno-depletion of cytosolic fractions (Figure 4). Note that the sensorgrams shown in (C) AP-3 resulted in a reduction of the binding activity to the LIMP-II wt and (D) represent the difference in binding to wt and mutant forms of tail that is comparable with that to the mutant tail peptide, indicating the tail peptides. In the latter, the critical tyrosine or leucine residues that AP-3 is the major cytosolic component accounting for the of the sorting signals had been substituted by alanine (Figure 1). cytosolic LIMP-II tail-binding activity. Tyrosinase, a melanosomal membrane protein, binds AP-3 AP-3 binding to lamp-1 and LAP It is known that most cells have specialized organelles The lysosomal membrane is enriched in a variety of other with features similar to that of lysosomes. One such membrane proteins that do not belong to the LIMP-II example is that of melanosomes, which play an important family. These lamp proteins, as well as the transmembrane role in pigmentation. A key protein of melanosomes is form of LAP, have in common the fact that their short tyrosinase, a membrane protein that is involved in melanine cytoplasmic tails contain a tyrosine-based sorting motif synthesis (Delmarmol and Beermann, 1996). Tyrosinase, which is critical for their targetting (Hunziker and Geuze, if transfected into COS cells, is targetted to lysosomes. In 1996). LAP has been shown to bind AP-2 but not AP-1 this context it should be noted that tyrosinase contains (Sosa et al., 1993), whereas lamp-1 is known to interact two di-leucine motifs in its cytoplasmic tail (Figure 1). with both AP-1 and AP-2 (Ho¨ning et al., 1996). Data However, it is not known which of the signals mediate regarding binding to AP-3 are missing. We therefore intracellular targetting of the protein. compared the binding of adaptors to peptides correspond- Since Drosophila AP-3 mutants show a dramatic loss ing with the cytoplasmic tails of LAP and lamp-1. In of pigmentation (Ooi et al., 1997; Simpson et al., 1997), contrast to LIMP-II (Figure 6), the interaction between it was of interest to analyze whether a tail peptide AP-3 and the tails of lamp-1 and LAP was of low corresponding to the wt tyrosinase tail binds to afﬁnity (Figure 9D). On the other hand, the interaction of adaptors. We therefore analyzed the ability of wt and lamp-1 and the LAP tail with a cytosolic fraction enriched mutant tyrosinase tail peptides (see Figure 1) to interact in AP-1 and AP-2 was of high afﬁnity (Figure 9C). The with AP-1, AP-2 and AP-3 enriched cytosolic fractions binding of AP-1/AP-2 to LAP and lamp-1 is dependent as described above. As shown in Figure 9B, we observed on the critical residues of their tyrosine-based sorting a high afﬁnity interaction between tyrosinase and AP-3. motifs, since binding to peptides in which the critical The relative equilibrium constant for the AP-3–tyrosinase tyrosine residues of the sorting motifs had been substituted interaction was 20% below the value obtained for the by alanines (see Figure 1) was close to background level LIMP-II tail–AP-3 interaction (arbitrarily set to 1). In (data not shown). The sensorgrams shown in Figure 9C contrast to the LIMP-II tail, the tyrosinase tail had a low and D have been corrected for background binding to the afﬁnity for AP-1/AP-2, which was 100 slower than mutant peptides. When puriﬁed AP-1 and AP-2 were used, the AP-1/AP-2–lamp-1 interaction (Figure 9A and C). In we calculated K values of 20 nM (LAP) and 64 nM order to test which of the two di-leucine motifs (or both) (lamp-1) (data not shown). As the concentration of AP-3 in the tail of tyrosinase is part of the sorting signal, we is unknown, the K value cannot be determined. However, also tested tyrosinase tail peptides for adaptor binding in the relative afﬁnity of AP-3 to LAP and lamp-1 was 25– which the proximal and distal di-leucine motifs were 1000 lower than that observed for AP-3 binding to mutated individually or in combination (Figure 1). Adaptor LIMP-II. Thus the interaction between AP-3 and lysosomal binding was totally abolished when both di-leucine motifs membrane proteins that contain tyrosine-based sorting were substituted for alanines. The same was observed if signals is of low afﬁnity. the proximal signal was destroyed. The tyrosinase tail 1310 LIMP-II tail and tyrosinase tail binding to AP3 peptide in which the distal di-leucine motif is mutated (Vijayasaradhi et al., 1995). It should be noted that exhibited a signiﬁcant residual afﬁnity to AP-3 (Figure tyrosinase contains a second potential di-leucine motif 9B). Thus the proximal tyrosinase di-leucine signal is located nine residues further towards the C-terminus essential for AP-3 binding in vitro. This result is consistent (Figure 1). The contribution of this second motif, which with in vivo experiments using gp75, a tyrosine-related is also preceded by acidic residues in positions –4 and protein that has a similar tail sequence. For gp75 it was –5, to sorting remains to be determined. However, the shown that a NQPLLTD sequence in the same position observation that a tyrosinase tail peptide, in which the as the relevant proximal tyrosinase motif is essential for second di-leucine pair is replaced by alanines, retains correct intracellular targetting (Vijayasaradhi et al., 1995). signiﬁcant binding for AP-3 makes it likely that the ﬁrst Substitution of either di-leucine motif abolished binding di-leucine motif is critical for sorting. of AP-1/AP-2 (Figure 9A). The experiments described The binding of AP-3 to tyrosinase and its likely above show, therefore, that the tyrosinase tail harbors a involvement in targetting of tyrosinase to melanosomes di-leucine-based sorting signal similar to that of LIMP-II, is in agreement with the observation that altered which mediates the high-afﬁnity interaction with AP-3. expression of garnet, the Drosophila ortholog of the mammalian δ-subunit of AP-3 (Ooi et al., 1997; Simpson et al., 1997), results in defects in eye pigmentation. This Discussion suggests that the pigmentation defect is produced by mis- Binding of AP-3 to the cytoplasmic tails of LIMP-II sorting of components of the biosynthetic machinery for and tyrosinase pigments, including tyrosinase, due to defective AP-3. The peptides corresponding to the cytoplasmic tail of LIMP-II or tyrosinase-bound AP-3 with high afﬁnity, Structural requirements for AP-3 binding while binding of cytosolic fractions enriched in AP-1 The μ3A and μ3B chains of AP-3 have been shown to and AP-2 was either below the limit of detection interact with tyrosine-based sorting signals in a yeast two- (LIMP-II) or of low afﬁnity (tyrosinase). The binding hybrid approach, in which the μ-chains were expressed of AP-3 to LIMP-II was characterized in more detail as fusions with the Gal-4 activation domain and the YQRL and shown to depend on leucine 18, which is a critical sorting signal contained in the itinerant Golgi protein component of the di-leucine-based motif involved in TGN38 in the context of the Gal-4 DNA binding the sorting of LIMP-II to lysosomes (Ogata and Fukuda, domains (Dell’Angelica et al., 1997b). In our system we 1994; Sandoval et al., 1994). Furthermore, binding was failed to observe any interaction of AP-3 with tail peptides greatly reduced by preincubating the LIMP-II-derived containing tyrosine-based sorting signals known to bind chip with an antibody which recognizes the LI motif AP-1 and/or AP-2 (lamp-1, LAP and MPR46). within the context of the C-terminal hexapeptide of the The only peptides with AP-3 binding activity were LIMP-II tail. For the binding studies, a cytosol fraction found to share a di-leucine-based sorting motif. It is, enriched in AP-3 and essentially free of AP-1 and AP-2 was however, of interest that the wt MPR46 tail peptide, which used. The complete loss of LIMP-II binding activity that bears a di-leucine-based signal that is critical for lysosomal was observed after two rounds of immuno-depletion of enzyme sorting (Johnson and Kornfeld, 1992), did not crude cytosol from AP-3 showed that AP-3 accounts for bind to AP-3. This observation suggests that the pairs of essentially all of the cytosolic binding activity to the LL- or LI- are not sufﬁcient for binding. Neighboring LIMP-II tail. The lack of puriﬁed AP-3 prevented us acidic residues in position –4 and –5 were of critical from calculating the equilibrium constant for binding. importance for the binding of the LIMP-II tail to AP-3. Furthermore, we cannot exclude that components com- A similar pair of acidic residues, in positions –4 and –5 plexed to AP-3 contribute to the observed biosensor relative to the di-leucine motif, is found also in the signal. tyrosinase tail (EEXXXLL) and the tail of invariant chain The cytoplasmic tails of LIMP-II and tyrosinase share (DDXXXLI). Since AP-3 does not bind to invariant a sorting motif of the di-leucine type (Ogata and chain (D.Rodionov, S.Honing, K.V.Figura and O.Bakke, Fukuda, 1994; Sandoval et al., 1994; Vijayasaradhi et al., unpublished), the sequence D/E EXXXLL/I is necessary, 1995) which in both is directly preceded by a D(E)ERXP but not sufﬁcient alone, for AP-3 binding. The speciﬁcity sequence. In this context, the acidic residues in positions of the AP-3–tail interaction must be determined by addi- –4 and –5 are critical for targetting of LIMP-II to tional structural features or unknown in vivo factors. It lysosomes in vivo (Pond et al., 1995; S.Martinez-Arca should be noted that in the MPR46-, MPR300- and CD3- and I.V.Sandoval, submitted). Here we have shown that γ chain cytoplasmic tails, acidic residues (single or pairs) the same residues are critical for binding of AP-3. Although are found neighboring the di-leucine motifs. Indeed, it has the functional signiﬁcance of the di-leucine motif for recently been shown for the CD3-γ chain that an aspartic sorting of tyrosinase remains to be demonstrated, two acid tail residue resembling a DXXXLL motif is important observations are of interest with regard to this. First, for the interaction with AP-1 and AP-2 (Dietrich et al., truncation of the cytoplasmic tail of tyrosinase, as it occurs 1997). This points to a general role of acidic residues in in mice with the platinum allele of tyrosinase at residue – adaptor binding to di-leucine motifs. 10 relative to the di-leucine motif, results in misrouting of tyrosinase and severe oculocutaneous albinism (Beermann Role of AP-3 in sorting to lysosomes and related et al., 1995). Secondly, the EXRQPLL heptamer sequence, melanosomes which is shared between tyrosinase and several other There is ample evidence that lysosomes and melanosomes melanosomal proteins, is critical for the sorting of the are related organelles; they share the internal acidic pH tyrosinase-related brown protein gp75 to melanosomes and membrane proteins such as lamp-1, LAP and other 1311 S.Ho¨ning, I.V.Sandoval and K.von Figura acidic hydrolases (Orlow et al., 1993; Schraermeyer, segregation of proteins that enter the late endosomal/ 1995). Moreover, the similarities extend to the protein lysosomal pathway from proteins such as invariant chain targetting machinery since melanosomal proteins, when and MPR46 which are targetted to the plasma membrane expressed in ﬁbroblasts, are transported to lysosomes or other intracellular organelles. The failure of lamp-1 and which together with the other observations has led to the LAP, which share with LIMP-II the lysosome as the ﬁnal view that melanosomes are specialized lysosomes (Orlow destination, to interact with AP-3 points to additional et al., 1995; Schraermeyer, 1995). The observation that routes from endosomes to lysosomes that are independent LIMP-II and tyrosinase share the interaction with AP-3 of AP-3. Only further detailed biochemical and morpholo- lends strong support to the view that AP-3 is involved in gical studies will help us to identify the intracellular sorting steps common to the biogenesis of lysosomes and location(s) at which AP-3 is functional in the pathways melanosomes. to lysosomes/melanosomes. The involvement of AP-3 in the transport of membrane proteins to lysosomes gains further support from a recent Materials and methods report about the functional role of AP-3 in yeast (Cowles et al., 1997a). The authors have ﬁrst identiﬁed the four Preparation of AP-1 and AP-2 yeast subunit homologs of AP-3 as APS3 (σ3A), APM3 The puriﬁcation and separation of the clathrin-associated protein com- plexes AP-1 and AP-2 were performed as described (Ho¨ning et al. 1997). (μ3A), APL5 (δ3) and APL6 (β3A), and show that yeast mutants lacking any of the four AP-3 subunits exhibit a Antibodies defect in the vacuolar sorting of alkaline phosphatase and The monoclonal antibodies against α-adaptin (clone 100/2) and γ-adaptin the vacuolar t-SNARE Vam3p. In addition, the transport (clone 100/3) were purchased from Sigma. The antiserum against the tail of LIMP-II was raised by the immunization of rabbits with a peptide defect in the AP-3 mutants is selective, as transport of corresponding to the last six tail amino acid residues (APLIRT) coupled other vacuolar proteins such as carboxypeptidase S and to hemocyanine-KLH. The antiserum obtained after six immunizations carboxypeptidase Y was not affected. In this context it recognized wt LIMP-II, but not a mutant form of LIMP-II in which should be noted that sorting of alkaline phosphatase to Leu18 of the cytoplasmic tail was replaced by Gly. The rabbit antibodies the yeast vacuole depends on a di-leucine-type signal in speciﬁc for the δ-subunit and the μ-subunit of the AP-3 complex were generously provided by Margaret Robinson and are described elsewhere its cytoplasmic tail (Cowles et al., 1997b). (Simpson et al. 1997). The question remains as to where in the cell the interaction between AP-3 and cargo proteins such as Preparation and fractionation of brain cytosol LIMP-II and tyrosinase occurs. Recent studies using Pig brain obtained from a local slaughterhouse was rinsed in 0.9% immunoﬂuorescence and immunogold labeling of NRK NaCl; surrounding tissue and blood vessels were removed before the brain was cut into small pieces. 1 g brain was homogenized in 2 ml cells have localized AP-3 to a late Golgi compartment buffer H (25 mM HEPES–KOH, pH 7.0, 125 mM K-acetate, 2.5 mM and to endosomal membranes (Newman et al., 1995; Mg-acetate, 1 mg/ml glucose, 0.1 mM EGTA, 1 μg/ml leupeptin, Dell’Angelica et al., 1997b; Simpson et al., 1997). It is 1 μg/ml pepstatin) 20 times using a dounce homogenizer. Subsequently, therefore likely that AP-3 function can be localized to the the homogenate was centrifuged at 4°C for 30 min at 100 000 g. The supernatant was collected and dialyzed twice for 4 h each time TGN, or an endosomal compartment or both. against buffer H. After ultracentrifugation as before, the supernatant We also performed double immunoﬂuorescence to co- was aliquoted and used in interaction analysis or for gel ﬁltration. localize AP-3 with different intracellular marker proteins, Alternatively, the brain cytosol was shock-frozen in liquid nitrogen including lamp-1, LIMP-II, TGN38, AP-1 and clathrin. for storage. However, we failed to detect a signiﬁcant overlap with Gel ﬁltration any of the markers used. It was evident that AP-3 is For gel ﬁltration experiments the brain cytosol (50 μl aliquots) was distributed in very small vesicular structures throughout passed over a Superdex-200 column connected to a SMART system the entire cell and not concentrated to a perinuclear (Pharmacia), equilibrated and eluted with buffer A (20 mM HEPES– compartment (data not shown). It should be noted, how- NaOH, pH 7.0, 150 mM NaCl, 10 mM KCl, 2 mM MgCl , 0.2 mM ever, that the steady-state distribution of proteins does not DTT) at a ﬂow-rate of 40 μl/min. Fractions of 50 μl were collected and analyzed by Western blot analysis. The proteins thyroglobulin (669 kDa), disclose their site of function. ferritin (440 kDa), catalase (232 kDa) and aldolase (158 kDa) from a A functional role of AP-3 at the level of the TGN is gel ﬁltration calibration kit (Pharmacia) were used as standards. supported by the observation that the binding of AP-3 to membranes is ARF1-dependent like that of the TGN- Production of brain cytosol depleted from AP-3 localized AP-1 (Simpson et al., 1997). Membrane proteins 100 μl of pig brain cytosol (5 mg/ml) were precleared with 20 μl S.aureus (Sigma) for 1 h at 4°C. Subsequently equal aliquots were that have been shown to interact with AP-1 include the incubated for 12 h with the antibody against the δ-subunit of AP-3, MPRs (Sosa et al., 1993; Mauxion et al., 1996, Glickman which was immobilized to S.aureus, or with S.aureus without antibody. et al., 1989), lamp-1 (Ho¨ning et al., 1996) and invariant After pelleting the S.aureus, the depletion step was repeated a second chain (Salamero et al., 1996). All these proteins are time. The ﬁnal supernatant was used for interaction analysis (see below). detected in early endosomes, and also to some extent at Peptide synthesis the plasma membrane. AP-3-mediated packaging of cargo All peptides used in this study were synthesized and puriﬁed as proteins at the TGN may, in contrast, initiate a pathway described (Ho¨ning et al., 1997). Their purity was conﬁrmed by HPLC, which bypasses the endosomal target of AP-1 dependent UV-spectrometry and mass-spectrometry. vesicles, resulting in a more direct delivery of proteins to SPR interaction analysis lysosomes (LIMP-II) or storage vesicles (tyrosinase). With The interaction between the different cytoplasmic tails and AP-1/AP-2 regard to this, the observation that LIMP-II is packed into or pig brain cytosolic fractions was analyzed in real-time by SPR coated vesicles at the TGN may be signiﬁcant (Barriocanal (Jonsson et al., 1991) using a BIAcore-2000 biosensor (Biacore AB). et al., 1986). An alternative model would allocate AP-3 The peptides were coupled to a CM5 sensor chip via their primary function to an endosomal compartment to function in the amino groups exactly according to the manufacturer’s instructions. 1312 LIMP-II tail and tyrosinase tail binding to AP3 Peptides that were available with an N-terminal cysteine residue were References coupled to the sensor chip using the thiol-coupling procedure. The Ahle,S., Mann,A., Eichelsbacher,U. and Ungewickell,E. (1988) Structural sensor chip surface is ﬁrst activated and derived to generate an active relationships between clathrin assembly proteins from the Golgi and disulﬁde on the chip surface. This group is then exchanged for the the plasma membrane. EMBO J., 7, 919–928. intrinsic thiol group of the peptide during peptide immobilization. Barriocanal,J., Bonifacino,J., Yuan,L. and Sandoval,I. (1986) Brieﬂy, the activation of the chip was carried out by the injection of Biosynthesis, glycosylation, movement through the golgi system, and 0.05 M N-hydroxysuccinimide/0.2 M N-ethyl-N’-dimethyl-aminopropyl- transport to lysosomes by an N-linked carbohydrate-independent carbodiimide for 4 min using a ﬂow-rate of 5 μl/min. The active mechanism of three lysosomal integral membrane proteins. J. Biol. disulﬁde was then introduced by a subsequent injection of 80 mM 2,2- Chem., 261, 16755–16763. pyridinyldithio-ethane-amine in 0.1 M borate buffer, pH 8.5, for 5 min. Beermann,F., Orlow,S.J., Boissy,R.E., Schmidt,A., Boissy,Y.L. and The immobilization was carried out by injecting the peptides at 0.1 mg/ml Lamoreux,M.L. (1995) Misrouting of tyrosinase with a truncated in 10 mM Na-acetate, pH 4.0, for 7 min. Excess reactive disulﬁdes and cytoplasmic tail as a result of the murine platinum [c(p)] mutation. non-covalently bound peptide were removed by a 5 min injection of 50 mM cysteine/1 M NaCl in 100 mM formate buffer, pH 4.3. Exp. Eye Res., 61, 599–607. All interaction experiments were performed with buffer A (see above) Boll,W., Gallusser,A. and Kirchhausen,T. (1995) Role of the regulatory at a ﬂow-rate of 20 μl/min unless otherwise stated. Association for 2 min domain of the EGF-receptor cytoplasmic tail in selective binding of was followed by dissociation for 2 min, during which buffer A was the clathrin-associated complex AP-2. Curr. Biol., 5, 1168–1178. perfused. A short pulse injection (15 s) of 20 mM NaOH/0.5% SDS Boll,W., Ohno,H., Zhou,S.Y., Rapoport,I., Cantley,L.C., Bonifacino,J.S. was used to regenerate the sensor chip surface after each experimental and Kirchhausen,T. (1996) Sequence requirements for the recognition cycle. The peptide-derived sensor chips remained stable and retained their of tyrosine-based endocytic signals by clathrin AP-2 complexes. speciﬁc binding capacity for 100 experimental cycles of association/ EMBO J., 15, 5789–5795. dissociation and regeneration. AP-1 and AP-2 were used at 100 nM; Cosson,P. and Letourneur,F. (1997) Coatomer (COPI)-coated vesicles: crude brain cytosol and the cytosolic fractions derived from gel ﬁltration role in intracellular transport and protein sorting. Curr. Opin. Cell (see above) were used at a ﬁnal protein concentration of ~50 μg/ml. Biol., 9, 484–487. Cowles,C., Odorizzi,G., Payne,G.S. and Emr,S.D. (1997a) The AP-3 Determination of kinetic rate constants complex is essential for cargo-selective transport to the yeast vacuole. The rate constants (k for association and k for dissociation) of the a d Cell, 91, 109–118. interaction between tail peptides and puriﬁed AP-1 or AP-2 were Cowles,C.R., Snyder,W.B., Burd,C.G. and Emr,S.D. (1997b) Novel calculated by using the evaluation software of the BIAcore 2000. The golgi to vacuole delivery pathway in yeast: identiﬁcation of a mathematical models that are used are described in more detail elsewhere sorting determinant and required transport component. EMBO J., 16, (O’Shannessy et al., 1993; Schuck and Minton 1996; Schuck, 1997). 2769–2782. Association was determined 15–20 s after switching from buffer ﬂow Dell’Angelica,E.C., Ooi,C.E. and Bonifacino,J.S. (1997a) Beta 3A- to adaptor solution to avoid distortions due to injection and mixing. The adaptin, a subunit of the adaptor-like complex AP-3. J. Biol. Chem., dissociation rate constants were determined 5–10 s after switching to 272, 15078–15084. buffer ﬂow. After a ﬁrst dissociation phase for ~30 s, further dissociation Dell’Angelica,E.C., Ohno,H., Ooi,C.E., Rabinovich,E., Roche,K.W. and of adaptors was very low. The association constant k , the dissociation Bonifacino,J.S. (1997b) AP-3: an adaptor-like protein complex with constant k and the calculation of the equilibrium constant K k /k d D d a ubiquitous expression. EMBO J., 16, 917–928. were determined by using the BIA evaluation software version 1.2, Delmarmol,V. and Beermann,F. (1996) Tyrosinase and related proteins assuming a ﬁrst order kinetic A B AB. The model used calculates in mammalian pigmentation. FEBS Lett., 381, 165–168. the association rate constant k and the steady state response level R a eq Denzer,K., Weber,B., Hillerehfeld,A., Vonﬁgura,K. and Pohlmann,R. by ﬁtting data to the equation: (1997) Identiﬁcation of three internalization sequences in the –(K C K )(t–t ) a n d 0 R R (1–e ) eq cytoplasmic tail of the 46 kDa mannose 6-phosphate receptor. Biochem. J., 326, 497–505. where t is the time in s; R , the steady state response level; and C, the eq Dietrich,J., Kastrup,J., Nielsen,B.L., Odum,N. and Geisler,C. (1997) molar concentration of adaptors in the injection solution. The steric Regulation and function of the CD3 gamma DxxxLL motif: a binding interference factor N, which describes the valency of the interaction site for adaptor protein-1 and adaptor protein-2 in vitro. J. Cell Biol., between the adaptors and the MPR46 tail, was set to 1. The dissociation 138, 271–281. rate constant k was determined by ﬁtting data to the equation: Glickman,J.A., Conibear,E. and Pearse,B.M.F. (1989) Speciﬁcity of –K (t–t ) d 0 R R e binding of clathrin adaptors to signals on the mannose-6-phosphate/ insulin-like growth factor II receptor. EMBO J., 8, 1041–1047. where R is the reponse level at the beginning time t of the dissociation 0 0 Gough,N. and Fambrough,D. (1997) Different steady state subcellular phase. This model, which has recently been applied to describe adaptor distributions of the three splice variants of lysosome-associated tail interaction (Heilker et al., 1996), is described in more detail elsewhere membrane protein lamp-2 are determined largely by the COOH- (Karlsson et al., 1991; O’Shannessy et al., 1993). It should be noted terminal amino acid residue. J. Cell Biol., 137, 1161–1169. that the models described above allow the determination of rate constants Heilker,R., Manning-Krieg,U., Zuber,J.F. and Spiess,M. (1996) In vitro without reaching equilibrium during the experimental cycle. The relative binding of clathrin adaptors to sorting signals correlates with rate constants given illustrate the afﬁnity differences between different tail peptides and are independent of adaptor concentration. endocytosis and basolateral sorting. EMBO J., 15, 2893–2899. Ho¨ning,S. and Hunziker,W. (1995) Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targetting of Electrophoresis and Western blotting The cytosolic fractions derived from gel ﬁltration were subjected to rat lgp120 (lamp-I) in MDCK cells. J. Cell Biol., 128, 321–332. SDS–PAGE analysis (Laemmli, 1970) and subsequently transferred onto Ho¨ning,S., Grifﬁth,J., Geuze,H.J. and Hunziker,W. (1996) The tyrosine- nitrocellulose membranes. The membranes were then probed for AP-2 based lysosomal targetting signal in lamp-1 mediates sorting into or AP-3 using the speciﬁc ﬁrst antibodies (see above) followed by golgi-derived clathrin-coated vesicles. EMBO J., 15, 5230–5239. horseradish peroxidase-labelled second antibodies, and detected by using Ho¨ning,S., Sosa,M., Hille-Rehfeld,A. and von Figura,K. (1997) The 46- the ECL system (Amersham). kDa mannose 6-phosphate receptor contains multiple binding sites for clathrin adaptors. J. Biol. Chem., 272, 19884–19890. Hunziker,W. and Geuze,H.J. (1996) Intracellular trafﬁcking of lysosomal Acknowledgements membrane proteins. BioEssays, 18, 379–389. Johnson,K.F. and Kornfeld,S. (1992) A His-Leu-Leu sequence near the We express our gratitude to Thomas C.Su¨dhof for the unlimited use of carboxyl terminus of the cytoplasmic domain of the cation-dependent his BIAcore and to Margaret Robinson for the gift of antibodies. The mannose 6-phosphate receptor is necessary for the lysosomal enzyme work of K.Neifer and B.Schmidt on peptide synthesis and puriﬁcation sorting function. J. Biol. Chem., 267, 17110–17115. is gratefully acknowledged. This work is supported by grants from Jonsson,U. et al. (1991) Real-time biospeciﬁc interaction analysis using the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen surface plasmon resonance and a sensor chip technology. Industrie to K.v.F., a grant of the Ministero de Ciencia y Cultura Espanol to I.V.S. and an EC grant (ERBFMAX-CT96-0058) to K.v.F. and I.V.S. Biotechniques, 11, 620–627. 1313 S.Ho¨ning, I.V.Sandoval and K.von Figura Karlsson,R., Michaelsson,A. and Mattsson,L. (1991) Kinetic analysis of tail, are critical for targetting of the resident lysosomal membrane protein monoclonal antibody–antigen interactions with a new biosensor based LIMP II to lysosomes. J. Biol. Chem., 269, 6622–6631. analytical system. J. Immun. Meth., 145, 229–240. Schraermeyer,U. (1995) Transport of endocytosed material into melanin Keen,J.H. (1987) Clathrin assembly proteins: afﬁnity puriﬁcation and a granules in cultured choroidal melanocytes of cattle—new insights into model for coat assembly. J. Cell Biol., 105, 1989–1998. the relationship of melanosomes with lysosomes. Pigm. Cell Res., 8, Keen,J.H., Willingham,M.C. and Pastan,I.H. (1979) Clathrin coated 209–214. vesicles: isolation, dissociation and factor-dependent reassociation of Schroder,S. and Ungewickell,E. (1991) Subunit interaction and function of clathrin-coated vesicle adaptors from the Golgi and the plasma clathrin baskets. Cell, 16, 303–312. membrane. J. Biol. Chem., 266, 7910–7918. Kirchhausen,T., Bonifacino,J.S. and Riezman,H. (1997) Linking cargo Schuck,P. (1997) Use of surface plasmon resonance to probe the to vesicle formation: receptor tail interactions with coat proteins. Curr. equilibrium and dynamic aspects of interactions between biological Opin. Cell Biol., 9, 488–495. macromolecules. Annu. Rev. Biophys. Biomol. Struc., 26, 541–566. Laemmli,U.K. (1970) Cleavage of structural proteins during assembly Schuck,P. and Minton,A.P. (1996) Analysis of mass transport-limited of the head of bacteriophage T4. Nature, 227, 680–685. binding kinetics in evanescent wave biosensors. Ann. Biochem., 240, Matter,K., Hunziker,W. and Mellman,I. (1992) Basolateral sorting of 262–272. LDL receptor in MDCK cells—the cytoplasmic domain contains 2 Simpson,F., Bright,N.A., West,M.A., Newman,L.S., Darnell,R.B. and Tyrosine-dependent targetting determinants. Cell, 71, 741–753. Robinson,M.S. (1996) A novel adaptor-related protein complex. J. Cell Matter,K., Yamamoto,E.M. and Mellman,I. (1994) Structural require- Biol., 133, 749–760. ments and sequence motifs for polarized sorting and endocytosis of Simpson,F., Peden,A.A., Christopoulou,L. and Robinson,M.S. (1997) LDL and fc receptors in MDCK cells. J. Cell Biol., 126, 991–1004. Characterization of the adaptor-related protein complex, AP-3. J. Cell Mauxion,F., LeBorgne,R., Munier-Lehmann,H. and Hoﬂack,B. (1996) Biol., 137, 835–845. A casein kinase II phosphorylation site in the cytoplasmic domain of Sosa,M.A., Schmidt,B., von Figura,K. and Hille-Rehfeld,A. (1993) In the cation-dependent mannose 6-phosphate receptor determines the vitro binding of plasma membrane-coated vesicle adaptors to the high afﬁnity interaction of the AP-1 golgi assembly proteins with cytoplasmic domain of lysosomal acid phosphatase. J. Biol. Chem., membranes. J. Biol. Chem., 271, 2171–2178. 268, 12537–12543. Newman,L.S., Mckeever,M.O., Okano,H.J. and Darnell,R.B. (1995) Stoorvogel,W., Oorschot,V. and Geuze,H.J. (1996) A novel class of Beta-NAP, a cerebellar degeneration antigen, is a neuron-speciﬁc clathrin-coated vesicles budding from endosomes. J. Cell Biol., 132, vesicle coat protein. Cell, 82, 773–783. 21–33. Ogata,S. and Fukuda,M. (1994) Lysosomal targetting of LIMP II Traub,L.M. (1997) Clathrin-associated adaptor proteins putting it all membrane glycoprotein requires a novel Leu-Ile motif at a particular together. Trends Cell Biol., 7, 43–46. position in its cytoplasmic tail. J. Biol. Chem., 269, 5210–5217. Vega,M., Segui-Real,B., Garcia,J., Cales,C., Rodriguez,F., Ohno,H. et al. (1995) Interaction of tyrosine-based sorting signals with Vanderkerckhove,J. and Sandoval,I. (1991) Cloning, sequencing, clathrin-associated proteins. Science, 269, 1872–1875. and expression of a cDNA encoding rat LIMP-II, a novel 74 kDa Ohno,H., Fournier,M.C., Poy,G. and Bonifacino,J.S. (1996) Structural lysosomal membrane protein related to the surface adhesion protein determinants of interaction of tyrosine-based sorting signals with the CD36. J. Biol. Chem., 266, 16818–16824. adaptor medium chains. J. Biol. Chem., 271, 29009–29015. Vijayasaradhi,S., Xu,Y.Q., Bouchard,B. and Houghton,A.N. (1995) Ooi,C.E., Moreira,J.E., Dell-Angelica,E.C., Poy,G., Wassarman,D.A. and Intracellular sorting and targetting of melanosomal membrane proteins: Bonifacino,J.S. (1997) Altered expression of a novel adaptin leads to identiﬁcation of signals for sorting of the human brown locus protein, defective pigment granule biogenesis in the drosophila eye color GP75. J. Cell Biol., 130, 807–820. mutant garnet. EMBO J., 16, 4508–4518. Wilde,A. and Brodsky,F.M. (1996) In vivo phosphorylation of adaptors Orlow,S.J. (1995) Melanosomes are specialized members of the regulates their interaction with clathrin. J. Cell Biol., 135, 635–645. lysosomal lineage of organelles. J. Invest. Dermatol., 105, 3–7. Orlow,S.J, Boissy,R.E., Moran,D.J. and Pifko-Hirst,S. (1993) Subcellular Received November 7, 1997; revised January 7, 1998; distribution of tyrosinase and tyrosinase-related protein-1: implications accepted January 8, 1998 for melanosomal biogenesis. J. Invest. Dermatol., 100, 55–64. O‘Shannessy,D., Brigham-Burk, M., Soneson,K., Hensley,P. and Brooks,I. (1993) Determination of rate and equilibrium constants for macromolecular interactions using surface plasmon resonance: Use of non linear least squares analysis methods. Ann. Biochem., 212, 457–468. Page,L.J. and Robinson,M.S. (1995) Targetting signals and subunit interactions in coated vesicle adaptor complexes. J. Cell Biol., 131, 619–630. Pearse,B.M.F. (1988) Receptors compete for adaptors found in plasma membrane coated pits. EMBO J., 7, 3331–3336. Pearse,B.M.F. and Robinson,M.S. (1990) Clathrin, adaptors and sorting. Annu. Rev. Cell Biol., 6, 151–171. Pond,L., Kuhn,L., Teyton,L., Schutze,M. and Peterson,P. (1995) A role for acidic residues in di-leucine motif-based targetting to the endocytosis pathway. J. Biol. Chem., 270, 19989–19997. Ponnambalam,S., Robinson,M.S., Jackson,A.P., Peiper,L. and Parham,P. (1990) Conservation and diversity in families of coated vesicles adaptins. J. Biol. Chem., 265, 4814–4820. Robinson,M.S. (1987) 100 kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies. J. Cell Biol., 104, 887–895. Robinson,M.S. (1997) Coats and vesicle budding. Trends Cell Biol., 7, 99–102. Salamero,J., LeBorgne,R., Saudrais,C., Goud,B. and Hoﬂack,B. (1996) Expression of major histocompatibility complex class II molecules in HeLa cells promotes the recruitment of AP-1 golgi-speciﬁc assembly proteins on golgi membranes. J. Biol. Chem., 271, 30318–30321. Sandoval,I.V. and Bakke,O. (1994) Targetting of membrane proteins to endosomes and lysosomes. Trends Cell Biol., 4, 292–297. Sandoval,I.V., Arredondo,J.J., Alcalde,J., Noriega,A.G., Vandekerckhov, J., Jimenez,M.A. and Rico,M. (1994) The residues Leu(Ile)(475)- Ile(Leu,Val,ALA)(476), contained in the extended carboxyl cytoplasmic http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals http://www.deepdyve.com/lp/springer-journals/a-di-leucine-based-motif-in-the-cytoplasmic-tail-of-limp-ii-and-a5d0V4Y7YR

Loading next page...

References (64)

B. Pearse, M. Robinson (1990)
Clathrin, adaptors, and sorting.
Annual review of cell biology, 6
Ulf Jonsson, L. Fägerstam, B. Ivarsson, B. Johnsson, R. Karlsson, K. Lundh, S. Löfås, B. Persson, H. Roos, I. Rönnberg (1991)
Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology.
BioTechniques, 11 5
H. Ohno, Jay Stewart, M. Fournier, H. Bosshart, Ina Rhee, S. Miyatake, T. Saito, A. Gallusser, T. Kirchhausen, J. Bonifacino (1995)
Interaction of tyrosine-based sorting signals with clathrin-associated proteins.
Science, 269 5232
U. Schraermeyer (1995)
Transport of endocytosed material into melanin granules in cultured choroidal melanocytes of cattle--new insights into the relationship of melanosomes with lysosomes.
Pigment cell research, 8 4
K. Matter, E. Yamamoto, I. Mellman (1994)
Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells
The Journal of Cell Biology, 126
J. Barriocanal, Juan, Bonifacino, Lydia Yuan, Ignacio, Sandoval (1986)
Biosynthesis, glycosylation, movement through the Golgi system, and transport to lysosomes by an N-linked carbohydrate-independent mechanism of three lysosomal integral membrane proteins.
The Journal of biological chemistry, 261 35
Robert Karlsson, Anne Michaelsson, Lars Mattsson (1991)
Kinetic analysis of monoclonal antibody-antigen interactions with a new biosensor based analytical system.
Journal of immunological methods, 145 1-2
D. O'Shannessy, M. Brigham-Burke, K. Soneson, P. Hensley, I. Brooks (1993)
Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: use of nonlinear least squares analysis methods.
Analytical biochemistry, 212 2
Lesley Page, M. Robinson (1995)
Targeting signals and subunit interactions in coated vesicle adaptor complexes
The Journal of Cell Biology, 131
M. Robinson (1987)
100-kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies
The Journal of Cell Biology, 104
T. Kirchhausen, J. Bonifacino, H. Riezman (1997)
Linking cargo to vesicle formation: receptor tail interactions with coat proteins.
Current opinion in cell biology, 9 4
M. Vega, B. Seguí‐Real, J. García, C. Cales, F. Rodríguez, J. Vanderkerckhove, I. Sandoval (1991)
Cloning, sequencing, and expression of a cDNA encoding rat LIMP II, a novel 74-kDa lysosomal membrane protein related to the surface adhesion protein CD36.
The Journal of biological chemistry, 266 25
Pierre Cosson, François Letourneur (1997)
Coatomer (COPI)-coated vesicles: role in intracellular transport and protein sorting.
Current opinion in cell biology, 9 4
J. Keen, M. Willingham, I. Pastan (1979)
Clathrin-coated vesicles: Isolation, dissociation and factor-dependent reassociation of clathrin baskets
Cell, 16
Karl Johnson, S. Kornfeld (1992)
A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function.
The Journal of biological chemistry, 267 24
Stefan Honing, Janice Griffith, H. Geuze, W. Hunziker (1996)
The tyrosine‐based lysosomal targeting signal in lamp‐1 mediates sorting into Golgi‐derived clathrin‐coated vesicles.
The EMBO Journal, 15
S. Orlow (1995)
Melanosomes are specialized members of the lysosomal lineage of organelles.
The Journal of investigative dermatology, 105 1
K. Denzer, B. Weber, A. Hille-Rehfeld, K. Figura, R. Pohlmann (1997)
Identification of three internalization sequences in the cytoplasmic tail of the 46 kDa mannose 6-phosphate receptor.
The Biochemical journal, 326 ( Pt 2)
W. Hunziker, H. Geuze (1996)
Intracellular trafficking of lysosomal membrane proteins
BioEssays, 18
W. Stoorvogel, V. Oorschot, H. Geuze (1996)
A novel class of clathrin-coated vesicles budding from endosomes
The Journal of Cell Biology, 132
D O'Shannessy, M Brigham‐Burk, K Soneson, P Hensley, I Brooks (1993)
Determination of rate and equilibrium constants for macromolecular interactions using surface plasmon resonance: Use of non linear least squares analysis methods
Ann Biochem, 212
Andrew Wilde, Frances Brodsky (1996)
In vivo phosphorylation of adaptors regulates their interaction with clathrin
The Journal of Cell Biology, 135
S. Orlow, R. Boissy, D. Moran, S. Pifko-Hirst (1993)
Subcellular distribution of tyrosinase and tyrosinase-related protein-1: implications for melanosomal biogenesis.
The Journal of investigative dermatology, 100 1
P. Schuck (1997)
Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules.
Annual review of biophysics and biomolecular structure, 26
S. Ahle, A. Mann, Uta Eichelsbacher, Ernst Ungewickell (1988)
Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane.
The EMBO Journal, 7
W. Boll, H. Ohno, Songyang Zhou, Iris Rapoport, L. Cantley, J. Bonifacino, T. Kirchhausen (1996)
Sequence requirements for the recognition of tyrosine‐based endocytic signals by clathrin AP‐2 complexes.
The EMBO Journal, 15
W. Boll, A. Gallusser, T. Kirchhausen (1995)
Role of the regulatory domain of the EGF-receptor cytoplasmic tail in selective binding of the clathrin-associated complex AP-2
Current Biology, 5
J. Salamero, R. Borgne, C. Saudrais, B. Goud, B. Hoflack (1996)
Expression of Major Histocompatibility Complex Class II Molecules in HeLa Cells Promotes the Recruitment of AP-1 Golgi-specific Assembly Proteins on Golgi Membranes*
The Journal of Biological Chemistry, 271
J. Keen (1987)
Clathrin assembly proteins: affinity purification and a model for coat assembly
The Journal of Cell Biology, 105
H. Ohno, M. Fournier, G. Poy, J. Bonifacino (1996)
Structural Determinants of Interaction of Tyrosine-based Sorting Signals with the Adaptor Medium Chains*
The Journal of Biological Chemistry, 271
R. Heilker, U. Manning-Krieg, Jean-Fran, ois Zuber, M. Spiess (1996)
In vitro binding of clathrin adaptors to sorting signals correlates with endocytosis and basolateral sorting.
The EMBO Journal, 15
P Schuck, AP Minton (1996)
Analysis of mass transport‐limited binding kinetics in evanescent wave biosensors
Ann Biochem, 240
N. Gough, D. Fambrough (1997)
Different Steady State Subcellular Distributions of the Three Splice Variants of Lysosome-associated Membrane Protein LAMP-2 Are Determined Largely by the COOH-terminal Amino Acid Residue
The Journal of Cell Biology, 137
J. Glickman, E. Conibear, B. Pearse (1989)
Specificity of binding of clathrin adaptors to signals on the mannose‐6‐phosphate/insulin‐like growth factor II receptor.
The EMBO Journal, 8
S. Höning, Walter Hunziker (1995)
Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targeting of rat lgp120 (lamp-I) in MDCK cells
The Journal of Cell Biology, 128
C. Cowles, G. Odorizzi, G. Payne, S. Emr (1997)
The AP-3 Adaptor Complex Is Essential for Cargo-Selective Transport to the Yeast Vacuole
Cell, 91
S. Ponnambalam, M. Robinson, A. Jackson, L. Peiperl, P. Parham (1990)
Conservation and diversity in families of coated vesicle adaptins.
The Journal of biological chemistry, 265 9
S. Ogata, Minoru Fukuda (1994)
Lysosomal targeting of Limp II membrane glycoprotein requires a novel Leu-Ile motif at a particular position in its cytoplasmic tail.
The Journal of biological chemistry, 269 7
F. Simpson, N. Bright, M West, L Newman, R Darnell, M. Robinson (1996)
A novel adaptor-related protein complex
The Journal of Cell Biology, 133
Friedrich Beermann, Seth Orlow, Raymond Boissy, Andrea Schmidt, Ying Boissy, M. Lamoreux (1995)
Misrouting of tyrosinase with a truncated cytoplasmic tail as a result of the murine platinum (cp) mutation.
Experimental eye research, 61 5
Ignacio Sandoval, J. Arredondo, J. Alcalde, A. Noriega, Joël Vandekerckhove, M. Jiménez, Manuel Rico (1994)
The residues Leu(Ile)475-Ile(Leu, Val, Ala)476, contained in the extended carboxyl cytoplasmic tail, are critical for targeting of the resident lysosomal membrane protein LIMP II to lysosomes.
The Journal of biological chemistry, 269 9
K. Matter, W. Hunziker, I. Mellman (1992)
Basolateral sorting of LDL receptor in MDCK cells: The cytoplasmic domain contains two tyrosine-dependent targeting determinants
Cell, 71
Chean Ooi, J. Moreira, E. Dell’Angelica, G. Poy, D. Wassarman, J. Bonifacino (1997)
Altered expression of a novel adaptin leads to defective pigment granule biogenesis in the Drosophila eye color mutant garnet
The EMBO Journal, 16
B. Pearse (1988)
Receptors compete for adaptors found in plasma membrane coated pits.
The EMBO Journal, 7
Tyrosine-dependent targetting determinants
F. Simpson, A. Peden, L. Christopoulou, M. Robinson (1997)
Characterization of the Adaptor-related Protein Complex, AP-3
The Journal of Cell Biology, 137
E. Dell’Angelica, C. Ooi, J. Bonifacino (1997)
β3A-adaptin, a Subunit of the Adaptor-like Complex AP-3*
The Journal of Biological Chemistry, 272
M. Sosa, B. Schmidt, K. Figura, A. Hille-Rehfeld (1993)
In vitro binding of plasma membrane-coated vesicle adaptors to the cytoplasmic domain of lysosomal acid phosphatase.
The Journal of biological chemistry, 268 17
L. Traub (1997)
Clathrin-associated adaptor proteins - putting it all together.
Trends in cell biology, 7 2
Sabrina Schröder, E. Ungewickell (1991)
Subunit interaction and function of clathrin-coated vesicle adaptors from the Golgi and the plasma membrane.
The Journal of biological chemistry, 266 12
V. Marmol, F. Beermann (1996)
Tyrosinase and related proteins in mammalian pigmentation
FEBS Letters, 381
P. Schuck, A. Minton (1996)
Analysis of mass transport-limited binding kinetics in evanescent wave biosensors.
Analytical biochemistry, 240 2
C. Cowles, W. Snyder, C. Burd, S. Emr (1997)
Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component
The EMBO Journal, 16
Leslie Pond, L. Kuhn, L. Teyton, M. Schutze, J. Tainer, M. Jackson, P. Peterson (1995)
A Role for Acidic Residues in Di-leucine Motif-based Targeting to the Endocytic Pathway (*)
The Journal of Biological Chemistry, 270
J. Dietrich, J. Kastrup, B. Nielsen, N. Ødum, C. Geisler (1997)
Regulation and Function of the CD3γ DxxxLL Motif: A Binding Site for Adaptor Protein-1 and Adaptor Protein-2 in Vitro
The Journal of Cell Biology, 138
S. Vijayasaradhi, Yiqing Xu, B. Bouchard, A. Houghton (1995)
Intracellular sorting and targeting of melanosomal membrane proteins: identification of signals for sorting of the human brown locus protein, gp75
The Journal of Cell Biology, 130
B. Pearse (1990)
Clathrin, Adapters, And Sorting
Annual Review of Cell and Developmental Biology, 6
S. Höning, M. Sosa, A. Hille-Rehfeld, K. Figura (1997)
The 46-kDa Mannose 6-Phosphate Receptor Contains Multiple Binding Sites for Clathrin Adaptors*
The Journal of Biological Chemistry, 272
E. Dell’Angelica, H. Ohno, C. Ooi, E. Rabinovich, K. Roche, J. Bonifacino (1997)
AP‐3: an adaptor‐like protein complex with ubiquitous expression
The EMBO Journal, 16
U. Laemmli (1970)
Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
Nature, 227
F. Mauxion, R. Borgne, H. Munier-Lehmann, B. Hoflack (1996)
A Casein Kinase II Phosphorylation Site in the Cytoplasmic Domain of the Cation-dependent Mannose 6-Phosphate Receptor Determines the High Affinity Interaction of the AP-1 Golgi Assembly Proteins with Membranes (*)
The Journal of Biological Chemistry, 271
Ignacio Sandoval, Oddmund Bakke (1994)
Targeting of membrane proteins to endosomes and lysosomes.
Trends in cell biology, 4 8
L. Newman, M. McKeever, H. Okano, R. Darnell (1995)
β-NAP, a cerebellar degeneration antigen, is a neuron-specific vesicle coat protein
Cell, 82
M. Robinson (1997)
Coats and vesicle budding.
Trends in cell biology, 7 3

Publisher: Springer Journals
Copyright: Copyright © European Molecular Biology Organization 1998
ISSN: 0261-4189
eISSN: 1460-2075
DOI: 10.1093/emboj/17.5.1304
Publisher site: See Article on Publisher Site

Abstract

The EMBO Journal Vol.17 No.5 pp.1304–1314, 1998 A di-leucine-based motif in the cytoplasmic tail of LIMP-II and tyrosinase mediates selective binding of AP-3 1 2 membrane proteins and its function is not yet understood Stefan Ho¨ning , Ignacio V.Sandoval and (Barrinocanal et al., 1986; Vega et al., 1991). Unlike the Kurt von Figura proteins of the lamp family, which contain a tyrosine- Institute for Biochemistry II, University of Go¨ttingen, Gosslerstr. 12d, based sorting signal of the GYXXZ type (Z being either 37073 Go¨ttingen, Germany and Centro de Biologia Molecular I, F, L or V) mediating intracellular sorting (Ho¨ning ‘Severo Ochoa’, Universidad Autonoma de Madrid, Madrid, Spain and Hunziker, 1995; Ho¨ning et al., 1996; Gough and Corresponding author Fambrough, 1997), the LIMP-II cytoplasmic tail harbors an LI signal of the di-leucine type. This signal has been Among the various coats involved in vesicular trans- shown to be necessary for the intracellular targetting of port, the clathrin associated coats that contain the the protein to lysosomes (Vega et al., 1991; Sandoval adaptor complexes AP-1 and AP-2 are the most et al., 1994). Though LIMP-II has been shown to be extensively characterized. The function of the recently packed into an as yet undeﬁned type of coated vesicles at described adaptor complex AP-3, which is similar to the TGN (Barriocanal et al., 1986), further details of the AP-1 and AP-2 in protein composition but does not intracellular pathways of LIMP-II are not known. associate with clathrin, is not known. By monitoring Clathrin-coated vesicles (CCVs) were the ﬁrst type surface plasmon resonance we observed that AP-3 is of intracellular vesicles to be identiﬁed and characterized. able to interact with the tail of the lysosomal integral Clathrin is the major structural component of CCVs; membrane protein LIMP-II and that this binding the speciﬁcity of its function is mediated by the depends on a DEXXXLI sequence in the LIMP-II tail. associated adaptor complexes AP-1 and AP-2 (reviewed Furthermore, AP-3 bound to the cytoplasmic tail of in Pearse and Robinson, 1990). Coated pits and coated the melanosome-associated protein tyrosinase which vesicles that are formed during endocytosis only contains a related EEXXXLL sequence. The tails of contain the AP-2 complex, while the same type of LIMP-II and tyrosinase either did not interact, or only coated pits and coated vesicles that derive from the interacted poorly, with AP-1 or AP-2. In contrast, the TGN contain the AP-1 complex (Robinson, 1987). The cytoplasmic tails of other membrane proteins con- four subunits that build up the AP-1 complex are the taining di-leucine and/or tyrosine-based sorting signals large γ- and β1-subunits together with the μ1-medium did not bind AP-3, but AP-1 and/or AP-2. This points and the σ1-small subunit. The related AP-2 complex to a function of AP-3 in intracellular sorting to lyso- consists of the α- and β2-subunits that assemble together somes and melanosomes of a subset of cargo proteins with the μ2- and σ2-subunits (Ahle et al., 1988; via di-leucine-based sorting motifs. Schro¨der and Ungewickell, 1991; Traub, 1997). The Keywords: biosensor/coated vesicles/endosome/ two complexes display signiﬁcant homology to each membrane trafﬁc/protein sorting other and in addition exhibit the similar structure of a large trunk that is separated from appendices (‘ears’) by a ﬂexible hinge. A major function of both adaptor complexes is to promote clathrin-lattice formation onto Introduction the respective membrane (Wilde and Brodsky, 1996). This function seems likely to involve the large subunits The lysosomal integral membrane protein LIMP-II γ- and β1 of AP-1 and α- and β2 of the AP-2 complex. (Barrinocanal et al., 1986; Vega et al., 1991), originally A second important function of AP-1 and AP-2 is the isolated from rat cells, belongs to a group of proteins that interaction with sorting signals that are present in the is highly enriched in lysosomal membranes. This group cytoplasmic tails of cargo proteins to be concentrated also comprises membrane proteins of the lamp-1, lamp-2 in CCVs (for review see Kirchhausen et al., 1997; and lamp-3 family, and lysosomal acid phosphatase (LAP) Robinson, 1997). Studies using different experimental (reviewed in Hunziker and Geuze, 1996). LIMP-II is a systems have revealed that tyrosine-based sorting signals type-III protein that traverses the membrane twice, with of membrane proteins such as the LDL receptor (Pearse, an N-terminal transmembrane anchor as a result of an 1988), LAP (Sosa et al., 1993), mutant hemagglutinin uncleaved signal peptide and a second membrane spanning (Heilker et al., 1996) and lamp-1 (Ho¨ning et al., 1996), region near the C-terminus, followed by a 20 amino acid as well as di-leucine-based sorting signals of membrane cytoplasmic tail. Most of the protein loops into the proteins such as the MPR300 (Glickman et al., 1989), lysosome where ﬁve cysteine residues may be involved mutant hemagglutinin (Heilker et al., 1996), invariant in disulﬁde bond formation. This region also contains 11 potential N-glycosylation sites. Glycosylation of the chain (Salamero et al., 1996) and MPR46 (Mauxion protein results in an apparent molecular weight of 74 kDa et al., 1996; Honing et al., 1997), can be recognized for the 477 amino acid mature protein. LIMP-II does by adaptor complexes. The situation could be more not display homology to any of the other lysosomal complex for proteins that contain more than one sorting 1304 © Oxford University Press LIMP-II tail and tyrosinase tail binding to AP3 signal in their tail, such as the LDL receptor (Matter Results et al., 1992, 1994) and the MPRs (Denzer et al., 1997; The LIMP-II tail is not recognized by AP-1 and AP-2 Ho¨ning et al., 1997), and it is not clear whether It has recently been shown that the lysosomal membrane adaptors recognize only a single motif or if they protein lamp-1 binds through a tyrosine-based sorting simultaneously interact with multiple sequence motifs. signal in its cytoplasmic tail to AP-1 and AP-2 with With the yeast two-hybrid system it was possible to high afﬁnity (Ho¨ning et al., 1996). In addition, electron show that different types of tyrosine-based sorting microscopic analysis of cells expressing wild-type (wt) or signals can be recognized by the μ-chains of AP-1 and mutant lamp-1 have shown a co-localization of lamp-1 AP-2 (Ohno et al., 1995, 1996; Boll et al., 1996). and AP-1, and the appearance of lamp-1 in TGN-derived However, it is still not clear if membrane proteins clathrin-coated vesicles. LIMP-II shares with lamp-1 the containing di-leucine-based sorting signals also interact direct routing to lysosomes, bypassing the cell surface with the μ-chain, or if one of the other subunits is (Vega et al., 1991; Sandoval et al., 1994). Unlike involved in the recognition of this type of sorting signal. lamp-1, the cytoplasmic tail of LIMP-II lacks a tyrosine- Apart from AP-1 and AP-2, which promote the forma- based sorting signal. Targetting of LIMP-II to lysosomes tion of clathrin-coated pits and vesicles at the plasma depends on a pair of LI residues which belongs to the membrane and at the TGN, many organelles do not seem class of di-leucine-based sorting motifs (Sandoval and to contain a clathrin coat yet vesicular transport to and Bakke, 1994; Sandoval et al., 1994). from all organelles is a prerequisite for the viability of To analyze a possible interaction of the LIMP-II cyto- plasmic tail with puriﬁed AP-1 and AP-2, we utilized a the living cell. This has led to the hypothesis that other biosensor system monitoring surface plasmon resonance protein coats beside clathrin may exist that have a similar (SPR). This method has been used in several studies to function in membrane trafﬁc (Stoorvogel et al., 1996; analyze the interaction between puriﬁed adaptors and the Robinson, 1997). The COP-I and COP-II coats that are EGF-receptor (Boll et al., 1995), the hemagglutinin tail involved in transport between the ER and the Golgi are (Heilker et al., 1996) and the lamp-1 tail (Ho¨ning et al., two examples of recently characterized non-clathrin coats 1996), and to deﬁne the AP-1 and AP-2 binding sites (for review see Cosson and Letourneur, 1997). The identi- on the MPR46 tail (Ho¨ning et al., 1997). A peptide ﬁcation of novel coats that may have a function in corresponding to the full-length LIMP-II tail (Figure 1) membrane protein sorting along their intracellular trafﬁc was synthesized, together with a mutant tail peptide where routes is a subject of active research (Stoorvogel et al., the leucine of the LI-sorting signal of LIMP-II is replaced 1996; Robinson, 1997). by glycine (LIMP-II L18G; Figure 1). This substitution Based on a signiﬁcant sequence homology to the known was previously shown to disrupt the lysosomal sorting of AP-1/AP-2 subunits and the search of EST databases, four LIMP-II (Sandoval et al., 1994). As a positive control we subunits of a novel adaptor-like complex named AP-3 used a peptide corresponding to the tail of MPR46, which have recently been cloned and characterized (Newman is known to bind to AP-1 and AP-2 (Sosa et al., 1993; et al., 1995; Dell’Angelica et al., 1997a; Simpson et al., Honing et al., 1997). The LIMP-II peptides were synthe- 1997). Like AP-1 and AP-2, AP-3 is a protein hetero- sized with an additional N-terminal cysteine residue which tetramer, which is composed of δ-adaptin (160 kDa), allows coupling of the peptides to the sensor chip surface β3-adaptin (140 kDa), the medium chain μ3 (47 kDa) and via this residue (see Materials and methods). All peptides the small chain σ3 (22 kDa). The complex is ubiquitously were coupled to a CM-5 sensor chip in equal densities expressed, with special variants of β3(β-NAP) and μ3 2 (~0.2 pmol/mm per peptide, data not shown) and tested (p47B) being expressed in brain. In contrast to AP-1 and for their ability to bind to puriﬁed AP-1 and AP-2. As AP-2, the AP-3 complex does not associate with clathrin. shown in Figure 2, only the MPR46 tail interacted with Immunoﬂuorescence analysis has revealed a distribution AP-1 (K 13 nM) and AP-2 (K 17 nM). In contrast, D D of AP-3 in the Golgi region and in peripheral structures neither the wt LIMP-II peptide nor the LIMP-II L18G that are thought to represent endosomes (Newman et al., peptide interacted detectably with AP-1 or AP-2 at 1995; Simpson et al., 1997). By the use of the yeast two- adaptor concentrations of up to 500 nM (shown for 100 nM hybrid system it was demonstrated that μ3 can interact AP-1 or AP-2, Figure 2). with tyrosine-based sorting signals similar to the clathrin- associated counterparts μ1 and μ2 in AP-1 and AP-2 Brain cytosol contains a component with LIMP-II (Dell’Angelica et al., 1997b). tail binding activity In an attempt to characterize the adaptor complexes Since we did not detect any binding of AP-1 or AP-2 to that bind to the cytoplasmic tail of LIMP-II, we observed the LIMP-II tail, we examined whether or not other that LIMP-II interacts with AP-3 but not with AP-1 or cytosolic components interact with the LIMP-II cyto- AP-2. This interaction is speciﬁc and dependent on the plasmic tail in a manner that is dependent on the LI LI motif that is critical for sorting of LIMP-II. In addition, sorting signal. For this purpose, cytosol from pig brain the acidic residues DE in positions –4 and –5 to the LI was prepared. When the brain cytosol was passed over signal appear to modulate this interaction. Furthermore, the MPR46 tail-derived sensor surface, a strong interaction the cytoplasmic tail of tyrosinase, a membrane protein of was observed suggesting the presence of functional lysosome-related melanosomes which contains a similar adaptors (see below and Figure 5). A similar interaction di-leucine motif, was also found to interact with AP-3. was monitored for the wt LIMP-II tail, while only a We propose a role of AP-3 in the sorting of a subset of reduced interaction was detected for the mutant LIMP- lysosomal and melanosomal membrane proteins. II L18G tail peptide (Figure 3). In the presence of 1305 S.Ho¨ning, I.V.Sandoval and K.von Figura Fig. 1. Amino acid sequences of cytoplasmic tail peptides. Peptides corresponding to the cytoplasmic tails of wt and mutant LIMP-II, lamp-1, LAP, tyrosinase and MPR46 are shown in the one letter code with the C-terminal end to the right. The open boxes on the left represent the junction of the membrane spanning region and the cytoplasmic tail. Residues known to be critical for sorting, and the corresponding mutations, are indicated by bold letters. All peptides represent the full-length cytoplasmic tails of the respective proteins except that of tyrosinase. The tyrosinase peptide lacks the membrane proximal residues 1–5. All peptides were immobilized on the sensor chip surface involving primary amino or thiol groups (see Materials and methods). carboxymethylated dextran, which minimizes unspeciﬁc binding to the chip surface which itself contains carboxy- methylated dextran, the residual binding to the mutant LIMP-II tail was negligible (Figure 3). In contrast, the signal obtained with the wt LIMP-II tail and the MPR46 tail peptide was only slightly reduced indicating the speciﬁcity of the interaction. Thus pig brain cytosol contains one or more components that have the ability to bind to the LIMP-II tail in a manner that is dependent on the LI sorting signal. Fig. 2. Binding of puriﬁed AP-1 and AP-2 to LIMP-II and MPR46 tail peptides. AP-1 and AP-2 preparations were passed over MPR46-, wt AP-3, but not AP-1 or AP-2, binds to the LIMP-II LIMP-II- and LIMP-II L18G-derived chip surfaces for 2 min tail (association) before the chip surface was washed with buffer A for The experiments described above demonstrated that a 2 min (dissociation). Only MPR46 was able to interact with both AP-1 and AP-2, whereas wt and mutant LIMP-II did not interact with either cytosolic factor(s) exists which can bind speciﬁcally to adaptor complex. the LIMP-II tail. To further analyze the cytosolic component(s) that binds to the cytoplasmic tails of MPR46 and LIMP-II, we fractionated pig brain cytosol LIMP-II from that binding to MPR46. Fractions 23–26, by gel ﬁltration. All fractions obtained were then while exhibiting an interaction with the MPR46 tail passed over sensor surfaces derived with the MPR46 peptide, did not interact at all with the LIMP-II peptides. tail, with wt LIMP-II or the LIMP-II L18G mutant tail On the other hand, fractions 28–30 exhibited binding peptide. To compare the binding, the resonance units activity for the LIMP-II tail but not for the MPR46 that remained bound to the chip-surface after a 2 min tail. Furthermore, the L18G mutant peptide was only association/2 min dissociation cycle were plotted. It poorly recognized by fractions 28–30. No other fractions should be noted that this experiment was performed besides those plotted in Figure 4 were found to bind without quenching non-speciﬁc interaction by adding to the tail peptides. carboxymethylated dextran. As shown in Figure 4, we As it had already been shown that MPR46 binds to observed a separation of the components that bind to both clathrin adaptors, it was suspected that fractions 1306 LIMP-II tail and tyrosinase tail binding to AP3 consistent with the ﬁnding that the LIMP-II tail does not interact with puriﬁed AP-1 or AP-2 (Figure 2). To further corroborate these ﬁndings, crude brain cytosol was passed over MPR46 and LIMP-II-derived surfaces for a 2 min association cycle. After a 2 min buffer wash (dissociation), antibodies that recognize native AP-2 or AP-3 were passed over the chip surface. As shown in Figure 5, when cytosol had been passed over the MPR46 tail-derived surface a strong signal was obtained with the anti-AP-2 antibody, indicating that AP-2 is recruited to the tail. In contrast, no binding of the anti-AP-3 antibody was observed, in agreement with the observation that AP-3 does not bind to MPR46. On the other hand, when crude brain cytosol was applied to the LIMP-II-derived surface a speciﬁc interaction with anti-AP-3 was observed, but not with anti-AP-2 (or anti- AP-1, data not shown). Thus the perfusion of adaptor- speciﬁc antibodies over the tail-derived chip surfaces that were ﬁrst allowed to recruit the adaptors from cytosol conﬁrmed that AP-3 binds to the LIMP-II tail. AP-3 binding to LIMP-II requires an acidic cluster in addition to the LI sorting motif It has previously been shown in COS cells transfected with tail-mutants of LIMP-II that the LI motif is critical for correct intracellular sorting. In addition, mutation of the aspartic and glutamic acidic residues at positions –5 and –4 relative to the LI motif also interferes with correct Fig. 3. Binding of pig brain cytosol to the LIMP-II cytosplasmic tail. Crude brain cytosol from pig brain was prepared and passed in the lysosomal targetting of LIMP-II (Sandoval et al., 1994; presence or absence of 1 mg/ml carboxymethylated dextran over tail- S.Martinez-Arca and I.V.Sandoval, submitted). In order to peptide derived sensor chip surfaces to record interaction. MPR46 and deﬁne further the sequence determinants in the LIMP-II wt LIMP-II were able to bind a component(s) of brain cytosol, tail that mediate AP-3 binding, a set of mutant tail peptides whereas binding to mutant LIMP-II L18G was of low afﬁnity. Carboxymethylated dextran, which reduces non-speciﬁc binding to the of LIMP-II (Figure 1) was synthesized to analyze their chip surface, abolished almost completely the residual binding to binding to AP-3 by SPR (see above and Figure 6). The LIMP-II L18G. importance of leucine 475 for AP-3 binding has already been shown since the control peptide of the LIMP-II tail, in which the leucine is replaced by glycine (LIMP-II 23–26 may contain AP-1 and AP-2. All fractions obtained L18G), does not bind AP-3 (Figures 3 and 4). Furthermore, from gel ﬁltration were subjected to SDS–PAGE and when a truncated tail-peptide of LIMP-II lacking the C- Western blotting, followed by incubation with anti- terminal pentapeptide PLIRT was immobilized, binding bodies speciﬁc for the known adaptor complexes. To of AP-3 was totally abolished (data not shown). This detect the AP-1 complex, a monoclonal antibody against result is in agreement with the data obtained from cells the γ-subunit was applied; the AP-2 complex was detected expressing the corresponding truncated LIMP-II protein by an α-subunit speciﬁc antibody; and the AP-3 complex which is delivered to the cell surface (Sandoval et al., was identiﬁed by antiserum speciﬁc for the δ-subunit 1994). (Simpson et al., 1997). As shown in Figure 4, the When the two amino acid residues DE, in positions AP-2 speciﬁc antibody only reacted with fractions 23–26. –4 and –5 relative to the LI motif, were mutated An identical pattern was observed with an antibody separately to alanines, a 20- to 60-fold decrease in against AP-1 (data not shown). In contrast, the anti-AP-3 afﬁnity to AP-3 as compared with the wt LIMP-II tail antibody reacted with fractions 28–30, but not with frac- was observed (Figure 6A). Moreover, the replacement tions 23–26. Thus, gel ﬁltration of brain cytosol leads to of both residues by arginine led to a 100-fold decrease the separation of AP-1/AP-2 from AP-3. The apparent in binding afﬁnity to AP-3. These results indicate that size of AP-3 as compared with a molecular weight both of the two acidic residues preceding the LI motif standard matched a molecular weight of ~350 kDa. In in the LIMP-II tail at positions –4 and –5 are necessary contrast, the clathrin-associated adaptor complexes for a high afﬁnity interaction with the AP-3 complex. appeared to run as a complex slightly larger than expected Another explanation would be that mutations within the from their calculated weight, which is comparable with tail peptide disrupt the structure of the immobilized that of AP-3. This unusual behaviour during gel ﬁltration peptide, thereby causing loss of adaptor binding. of AP-1 and AP-2 has been noted previously (Keen To assess the plausibility of this assumption, three et al., 1979; Keen 1987). Most interestingly, the LIMP-II different mutant LIMP-II tail peptides were synthesized tail is only recognized by factors present in the AP-3 (LIMP-II S6A, E9Q and T11A) which correspond to enriched fractions but does not show any interaction with LIMP-II mutants that are known to be correctly sorted AP-1/AP-2-containing fractions (Figure 4). This result is in vivo (Sandoval et al., 1994). As shown in Figure 1307 S.Ho¨ning, I.V.Sandoval and K.von Figura Fig. 4. Gel ﬁltration analysis of pig brain cytosol. Cytosol from pig brain was fractionated by gel ﬁltration on Superdex-200. The fractions were analyzed by SDS–PAGE and Western blotting, and probed with antibodies to the α-subunit of AP-2 and the δ-subunit of AP-3 as indicated in the ﬁgure. Furthermore, each fraction was tested for interaction with the MPR46, wt LIMP-II and LIMP-II L18G-derived sensor chip surfaces. The bars represent the resonance units that remained associated to the chip after an association (2 min)/dissociation (2 min) cycle. No other fraction, apart from those indicated, exhibited any interaction with the tail peptides. The values presented are corrected for background binding which was below 55 RU in all fractions. 6B, the observed binding of AP-3 to these mutant tail peptides was nearly identical to that observed for wt LIMP-II, indicating that mutations within the tail peptide do not lead a priori to loss of adaptor binding. In addition, previous NMR analysis of the LIMP-II tail peptide (Sandoval et al., 1994) has revealed the predominance of random coil conformations indicating a high ﬂexibility of the LIMP-II tail. Taken together, the data obtained with the biosensor conﬁrm the in vivo data showing that sorting of LIMP-II is dependent on a di-leucine motif which functions in the context of two acidic residues. The observation that substitutions of several residues known not to interfere with sorting of LIMP-II also do not affect the interaction of the LIMP-II tail with AP-3 underlines the signiﬁcance of the in vitro data. The speciﬁcity of the AP-3 LIMP-II tail interaction was Fig. 5. AP-3 is the cytosolic component that binds to the LIMP-II tail peptide. A cytosol fraction enriched in AP-3 was passed over a LIMP- also conﬁrmed by the perfusion of the LIMP-II-derived II-derived chip surface (left) and an AP-2 enriched cytosol fraction chip with an anti-LIMP-II tail antiserum prior to the over a MPR46-derived chip surface (right). After an association/ injection of the AP-3 enriched fraction. The antiserum, dissociation cycle, antibodies speciﬁc for AP-3 (δ-subunit) or AP-2 which speciﬁcally recognizes the C-terminal 6 amino acids (α-subunit) were injected. Note that LIMP-II is recruiting AP-3 but not AP-2 from the cytosol, as revealed by the positive signal obtained of the LIMP-II tail, was injected at a low ﬂow-rate to by the anti-AP-3 antibody. On the other hand, the MPR46 recruits obtain maximal binding. Subsequently, an AP-3-enriched AP-2 but not AP-3 from the cytosol, as indicated by the strong signal fraction (fraction 29, see Figure 4) was passed over the with the anti-AP-2 antibody. surface. The antibody perfusion led to a 70% loss in binding of AP-3, while perfusion with a control serum did not interfere with the subsequent binding of AP-3 to the LIMP-II tail (Figure 7). It should be noted that the tail-speciﬁc antiserum to a hexapeptide comprising the LIMP-II tail antibody does not cross-react with the mutant LI-sorting motif leads to the inhibition of AP-3 interaction. tail-peptide L18G (data not shown). Thus binding of the Taken together the experiments described above demon- 1308 LIMP-II tail and tyrosinase tail binding to AP3 Fig. 7. Binding of AP-3 to the LIMP-II tail is sensitive to the injection of anti-LIMP-II tail antiserum. An anti-LIMP-II tail antiserum or a control serum was passed over the LIMP-II-derived chip. To obtain maximum binding of the antiserum to the tail-peptide the ﬂow-rate was reduced to 5 μl/min. After antibody washing was extended to 6 min to reach a near constant resonance, an AP-3 enriched cytosol fraction (black curve, see Figure 4) was passed over the chip surface, followed by a wash with buffer. As a control, the dissociation of the Fig. 6. Acidic residues preceding the LI motif in the LIMP-II tail are antibodies from the LIMP-II tail peptide was recorded separately for necessary for high afﬁnity binding of AP-3. LIMP-II tail peptides with up to 10 min (grey curve). Note that AP-3 binding following mutations of the acidic residues D12 and/or E13 (Figure 1) were 2 saturation of the chip surface with the anti-LIMP-II tail antiserum (A) coupled to the CM-5 sensor chip in equal densities (0.2 pmol/mm was reduced by 70% of the control (B). 10%). The derived surfaces were then tested for their ability to interact with an AP-3 enriched cytosol fraction (A). Tail peptides that correspond to LIMP-II mutants known not to interfere with sorting in vivo served as controls (B). The kinetic values for the on-rate (k ), bearing Staphylococcus aureus cells. Control cytosol was the off-rate (k ) and the equilibrium constant K (k /k ) represent d D a d treated likewise except that the anti-AP-3 antiserum was relative afﬁnities to that of the wt LIMP-II tail set to 1 (C). omitted (see Materials and methods). Identical aliquots of immuno-depleted and control cytosol were then analyzed for binding to the wt LIMP-II as well as to the mutant strate that a DEXXXLI-sorting motif in the LIMP-II tail LIMP-II tail, and to the MPR46 tail. As shown in Figure mediates a high afﬁnity interaction with AP-3. 8, after the incubation without antibody the cytosol retained 80% of its original binding activity as revealed by incuba- AP-3 is the major cytosolic factor that has a tion with the LIMP-II and the MPR46 tail peptides. LIMP-II binding capacity However, if the cytosol was depleted from AP-3 by The experiments described above provide evidence that incubation with the speciﬁc antibody prior to SPR analysis, the AP-3 complex becomes bound to the cytoplasmic tail binding of the cytosol to the wt LIMP-II tail was reduced of LIMP-II. However, it is possible that additional cytosolic by 75%. The residual binding is in the same range as factors contribute to the binding activity detected by SPR that observed for the binding of immuno-depleted or and that binding of AP-3 may even be of an indirect control cytosol to the L18G mutant tail-peptide. It nature. To test this possibility, cytosol was immuno- should be noted that immuno-depleted cytosol retains its depleted of AP-3. If cytosolic factors apart from AP-3 ability to bind to the MPR46 tail, indicating the speciﬁc bind to the LIMP-II tail, immuno-depletion of AP-3 from depletion of AP-3. These results indicate that AP-3 cytsosol should only partially reduce the binding activity. The brain cytosol was depleted from AP-3 by two rounds accounts for essentially all of the speciﬁc binding activity of incubation with anti-AP-3 antiserum and protein A, to the LIMP-II tail as detected by SPR. 1309 S.Ho¨ning, I.V.Sandoval and K.von Figura Fig. 8. Cytosol depleted from AP-3 loses the LIMP-II tail binding activity. Crude brain cytosol was precleared by incubation for 1 h with S.aureus followed by centrifugation. The supernatant was divided into three aliquots. One was used directly for SPR analysis (open bars). The other aliquots were subjected to two cycles of immuno-depletion with anti-AP-3 antiserum pre-adsorbed to S.aureus (ﬁlled bars) or Fig. 9. Tail peptides that can interact with AP-3 in vitro. The wt and S.aureus alone (hatched bars) prior to SPR analysis (see Materials and mutant tail peptides of tyrosinase (A and B), LAP and lamp-1 (C and methods). Binding activity of pre-cleared cytosol to the LIMP-II tail or D) were immobilized on the sensor chip and analyzed for their binding to the MPR46 tail (determined as in Figure 4) was set to 100% and capacity of AP-1/AP-2 (A and C) and AP-3 (B and D) enriched used as reference for the other binding activities. Immuno-depletion of cytosolic fractions (Figure 4). Note that the sensorgrams shown in (C) AP-3 resulted in a reduction of the binding activity to the LIMP-II wt and (D) represent the difference in binding to wt and mutant forms of tail that is comparable with that to the mutant tail peptide, indicating the tail peptides. In the latter, the critical tyrosine or leucine residues that AP-3 is the major cytosolic component accounting for the of the sorting signals had been substituted by alanine (Figure 1). cytosolic LIMP-II tail-binding activity. Tyrosinase, a melanosomal membrane protein, binds AP-3 AP-3 binding to lamp-1 and LAP It is known that most cells have specialized organelles The lysosomal membrane is enriched in a variety of other with features similar to that of lysosomes. One such membrane proteins that do not belong to the LIMP-II example is that of melanosomes, which play an important family. These lamp proteins, as well as the transmembrane role in pigmentation. A key protein of melanosomes is form of LAP, have in common the fact that their short tyrosinase, a membrane protein that is involved in melanine cytoplasmic tails contain a tyrosine-based sorting motif synthesis (Delmarmol and Beermann, 1996). Tyrosinase, which is critical for their targetting (Hunziker and Geuze, if transfected into COS cells, is targetted to lysosomes. In 1996). LAP has been shown to bind AP-2 but not AP-1 this context it should be noted that tyrosinase contains (Sosa et al., 1993), whereas lamp-1 is known to interact two di-leucine motifs in its cytoplasmic tail (Figure 1). with both AP-1 and AP-2 (Ho¨ning et al., 1996). Data However, it is not known which of the signals mediate regarding binding to AP-3 are missing. We therefore intracellular targetting of the protein. compared the binding of adaptors to peptides correspond- Since Drosophila AP-3 mutants show a dramatic loss ing with the cytoplasmic tails of LAP and lamp-1. In of pigmentation (Ooi et al., 1997; Simpson et al., 1997), contrast to LIMP-II (Figure 6), the interaction between it was of interest to analyze whether a tail peptide AP-3 and the tails of lamp-1 and LAP was of low corresponding to the wt tyrosinase tail binds to afﬁnity (Figure 9D). On the other hand, the interaction of adaptors. We therefore analyzed the ability of wt and lamp-1 and the LAP tail with a cytosolic fraction enriched mutant tyrosinase tail peptides (see Figure 1) to interact in AP-1 and AP-2 was of high afﬁnity (Figure 9C). The with AP-1, AP-2 and AP-3 enriched cytosolic fractions binding of AP-1/AP-2 to LAP and lamp-1 is dependent as described above. As shown in Figure 9B, we observed on the critical residues of their tyrosine-based sorting a high afﬁnity interaction between tyrosinase and AP-3. motifs, since binding to peptides in which the critical The relative equilibrium constant for the AP-3–tyrosinase tyrosine residues of the sorting motifs had been substituted interaction was 20% below the value obtained for the by alanines (see Figure 1) was close to background level LIMP-II tail–AP-3 interaction (arbitrarily set to 1). In (data not shown). The sensorgrams shown in Figure 9C contrast to the LIMP-II tail, the tyrosinase tail had a low and D have been corrected for background binding to the afﬁnity for AP-1/AP-2, which was 100 slower than mutant peptides. When puriﬁed AP-1 and AP-2 were used, the AP-1/AP-2–lamp-1 interaction (Figure 9A and C). In we calculated K values of 20 nM (LAP) and 64 nM order to test which of the two di-leucine motifs (or both) (lamp-1) (data not shown). As the concentration of AP-3 in the tail of tyrosinase is part of the sorting signal, we is unknown, the K value cannot be determined. However, also tested tyrosinase tail peptides for adaptor binding in the relative afﬁnity of AP-3 to LAP and lamp-1 was 25– which the proximal and distal di-leucine motifs were 1000 lower than that observed for AP-3 binding to mutated individually or in combination (Figure 1). Adaptor LIMP-II. Thus the interaction between AP-3 and lysosomal binding was totally abolished when both di-leucine motifs membrane proteins that contain tyrosine-based sorting were substituted for alanines. The same was observed if signals is of low afﬁnity. the proximal signal was destroyed. The tyrosinase tail 1310 LIMP-II tail and tyrosinase tail binding to AP3 peptide in which the distal di-leucine motif is mutated (Vijayasaradhi et al., 1995). It should be noted that exhibited a signiﬁcant residual afﬁnity to AP-3 (Figure tyrosinase contains a second potential di-leucine motif 9B). Thus the proximal tyrosinase di-leucine signal is located nine residues further towards the C-terminus essential for AP-3 binding in vitro. This result is consistent (Figure 1). The contribution of this second motif, which with in vivo experiments using gp75, a tyrosine-related is also preceded by acidic residues in positions –4 and protein that has a similar tail sequence. For gp75 it was –5, to sorting remains to be determined. However, the shown that a NQPLLTD sequence in the same position observation that a tyrosinase tail peptide, in which the as the relevant proximal tyrosinase motif is essential for second di-leucine pair is replaced by alanines, retains correct intracellular targetting (Vijayasaradhi et al., 1995). signiﬁcant binding for AP-3 makes it likely that the ﬁrst Substitution of either di-leucine motif abolished binding di-leucine motif is critical for sorting. of AP-1/AP-2 (Figure 9A). The experiments described The binding of AP-3 to tyrosinase and its likely above show, therefore, that the tyrosinase tail harbors a involvement in targetting of tyrosinase to melanosomes di-leucine-based sorting signal similar to that of LIMP-II, is in agreement with the observation that altered which mediates the high-afﬁnity interaction with AP-3. expression of garnet, the Drosophila ortholog of the mammalian δ-subunit of AP-3 (Ooi et al., 1997; Simpson et al., 1997), results in defects in eye pigmentation. This Discussion suggests that the pigmentation defect is produced by mis- Binding of AP-3 to the cytoplasmic tails of LIMP-II sorting of components of the biosynthetic machinery for and tyrosinase pigments, including tyrosinase, due to defective AP-3. The peptides corresponding to the cytoplasmic tail of LIMP-II or tyrosinase-bound AP-3 with high afﬁnity, Structural requirements for AP-3 binding while binding of cytosolic fractions enriched in AP-1 The μ3A and μ3B chains of AP-3 have been shown to and AP-2 was either below the limit of detection interact with tyrosine-based sorting signals in a yeast two- (LIMP-II) or of low afﬁnity (tyrosinase). The binding hybrid approach, in which the μ-chains were expressed of AP-3 to LIMP-II was characterized in more detail as fusions with the Gal-4 activation domain and the YQRL and shown to depend on leucine 18, which is a critical sorting signal contained in the itinerant Golgi protein component of the di-leucine-based motif involved in TGN38 in the context of the Gal-4 DNA binding the sorting of LIMP-II to lysosomes (Ogata and Fukuda, domains (Dell’Angelica et al., 1997b). In our system we 1994; Sandoval et al., 1994). Furthermore, binding was failed to observe any interaction of AP-3 with tail peptides greatly reduced by preincubating the LIMP-II-derived containing tyrosine-based sorting signals known to bind chip with an antibody which recognizes the LI motif AP-1 and/or AP-2 (lamp-1, LAP and MPR46). within the context of the C-terminal hexapeptide of the The only peptides with AP-3 binding activity were LIMP-II tail. For the binding studies, a cytosol fraction found to share a di-leucine-based sorting motif. It is, enriched in AP-3 and essentially free of AP-1 and AP-2 was however, of interest that the wt MPR46 tail peptide, which used. The complete loss of LIMP-II binding activity that bears a di-leucine-based signal that is critical for lysosomal was observed after two rounds of immuno-depletion of enzyme sorting (Johnson and Kornfeld, 1992), did not crude cytosol from AP-3 showed that AP-3 accounts for bind to AP-3. This observation suggests that the pairs of essentially all of the cytosolic binding activity to the LL- or LI- are not sufﬁcient for binding. Neighboring LIMP-II tail. The lack of puriﬁed AP-3 prevented us acidic residues in position –4 and –5 were of critical from calculating the equilibrium constant for binding. importance for the binding of the LIMP-II tail to AP-3. Furthermore, we cannot exclude that components com- A similar pair of acidic residues, in positions –4 and –5 plexed to AP-3 contribute to the observed biosensor relative to the di-leucine motif, is found also in the signal. tyrosinase tail (EEXXXLL) and the tail of invariant chain The cytoplasmic tails of LIMP-II and tyrosinase share (DDXXXLI). Since AP-3 does not bind to invariant a sorting motif of the di-leucine type (Ogata and chain (D.Rodionov, S.Honing, K.V.Figura and O.Bakke, Fukuda, 1994; Sandoval et al., 1994; Vijayasaradhi et al., unpublished), the sequence D/E EXXXLL/I is necessary, 1995) which in both is directly preceded by a D(E)ERXP but not sufﬁcient alone, for AP-3 binding. The speciﬁcity sequence. In this context, the acidic residues in positions of the AP-3–tail interaction must be determined by addi- –4 and –5 are critical for targetting of LIMP-II to tional structural features or unknown in vivo factors. It lysosomes in vivo (Pond et al., 1995; S.Martinez-Arca should be noted that in the MPR46-, MPR300- and CD3- and I.V.Sandoval, submitted). Here we have shown that γ chain cytoplasmic tails, acidic residues (single or pairs) the same residues are critical for binding of AP-3. Although are found neighboring the di-leucine motifs. Indeed, it has the functional signiﬁcance of the di-leucine motif for recently been shown for the CD3-γ chain that an aspartic sorting of tyrosinase remains to be demonstrated, two acid tail residue resembling a DXXXLL motif is important observations are of interest with regard to this. First, for the interaction with AP-1 and AP-2 (Dietrich et al., truncation of the cytoplasmic tail of tyrosinase, as it occurs 1997). This points to a general role of acidic residues in in mice with the platinum allele of tyrosinase at residue – adaptor binding to di-leucine motifs. 10 relative to the di-leucine motif, results in misrouting of tyrosinase and severe oculocutaneous albinism (Beermann Role of AP-3 in sorting to lysosomes and related et al., 1995). Secondly, the EXRQPLL heptamer sequence, melanosomes which is shared between tyrosinase and several other There is ample evidence that lysosomes and melanosomes melanosomal proteins, is critical for the sorting of the are related organelles; they share the internal acidic pH tyrosinase-related brown protein gp75 to melanosomes and membrane proteins such as lamp-1, LAP and other 1311 S.Ho¨ning, I.V.Sandoval and K.von Figura acidic hydrolases (Orlow et al., 1993; Schraermeyer, segregation of proteins that enter the late endosomal/ 1995). Moreover, the similarities extend to the protein lysosomal pathway from proteins such as invariant chain targetting machinery since melanosomal proteins, when and MPR46 which are targetted to the plasma membrane expressed in ﬁbroblasts, are transported to lysosomes or other intracellular organelles. The failure of lamp-1 and which together with the other observations has led to the LAP, which share with LIMP-II the lysosome as the ﬁnal view that melanosomes are specialized lysosomes (Orlow destination, to interact with AP-3 points to additional et al., 1995; Schraermeyer, 1995). The observation that routes from endosomes to lysosomes that are independent LIMP-II and tyrosinase share the interaction with AP-3 of AP-3. Only further detailed biochemical and morpholo- lends strong support to the view that AP-3 is involved in gical studies will help us to identify the intracellular sorting steps common to the biogenesis of lysosomes and location(s) at which AP-3 is functional in the pathways melanosomes. to lysosomes/melanosomes. The involvement of AP-3 in the transport of membrane proteins to lysosomes gains further support from a recent Materials and methods report about the functional role of AP-3 in yeast (Cowles et al., 1997a). The authors have ﬁrst identiﬁed the four Preparation of AP-1 and AP-2 yeast subunit homologs of AP-3 as APS3 (σ3A), APM3 The puriﬁcation and separation of the clathrin-associated protein com- plexes AP-1 and AP-2 were performed as described (Ho¨ning et al. 1997). (μ3A), APL5 (δ3) and APL6 (β3A), and show that yeast mutants lacking any of the four AP-3 subunits exhibit a Antibodies defect in the vacuolar sorting of alkaline phosphatase and The monoclonal antibodies against α-adaptin (clone 100/2) and γ-adaptin the vacuolar t-SNARE Vam3p. In addition, the transport (clone 100/3) were purchased from Sigma. The antiserum against the tail of LIMP-II was raised by the immunization of rabbits with a peptide defect in the AP-3 mutants is selective, as transport of corresponding to the last six tail amino acid residues (APLIRT) coupled other vacuolar proteins such as carboxypeptidase S and to hemocyanine-KLH. The antiserum obtained after six immunizations carboxypeptidase Y was not affected. In this context it recognized wt LIMP-II, but not a mutant form of LIMP-II in which should be noted that sorting of alkaline phosphatase to Leu18 of the cytoplasmic tail was replaced by Gly. The rabbit antibodies the yeast vacuole depends on a di-leucine-type signal in speciﬁc for the δ-subunit and the μ-subunit of the AP-3 complex were generously provided by Margaret Robinson and are described elsewhere its cytoplasmic tail (Cowles et al., 1997b). (Simpson et al. 1997). The question remains as to where in the cell the interaction between AP-3 and cargo proteins such as Preparation and fractionation of brain cytosol LIMP-II and tyrosinase occurs. Recent studies using Pig brain obtained from a local slaughterhouse was rinsed in 0.9% immunoﬂuorescence and immunogold labeling of NRK NaCl; surrounding tissue and blood vessels were removed before the brain was cut into small pieces. 1 g brain was homogenized in 2 ml cells have localized AP-3 to a late Golgi compartment buffer H (25 mM HEPES–KOH, pH 7.0, 125 mM K-acetate, 2.5 mM and to endosomal membranes (Newman et al., 1995; Mg-acetate, 1 mg/ml glucose, 0.1 mM EGTA, 1 μg/ml leupeptin, Dell’Angelica et al., 1997b; Simpson et al., 1997). It is 1 μg/ml pepstatin) 20 times using a dounce homogenizer. Subsequently, therefore likely that AP-3 function can be localized to the the homogenate was centrifuged at 4°C for 30 min at 100 000 g. The supernatant was collected and dialyzed twice for 4 h each time TGN, or an endosomal compartment or both. against buffer H. After ultracentrifugation as before, the supernatant We also performed double immunoﬂuorescence to co- was aliquoted and used in interaction analysis or for gel ﬁltration. localize AP-3 with different intracellular marker proteins, Alternatively, the brain cytosol was shock-frozen in liquid nitrogen including lamp-1, LIMP-II, TGN38, AP-1 and clathrin. for storage. However, we failed to detect a signiﬁcant overlap with Gel ﬁltration any of the markers used. It was evident that AP-3 is For gel ﬁltration experiments the brain cytosol (50 μl aliquots) was distributed in very small vesicular structures throughout passed over a Superdex-200 column connected to a SMART system the entire cell and not concentrated to a perinuclear (Pharmacia), equilibrated and eluted with buffer A (20 mM HEPES– compartment (data not shown). It should be noted, how- NaOH, pH 7.0, 150 mM NaCl, 10 mM KCl, 2 mM MgCl , 0.2 mM ever, that the steady-state distribution of proteins does not DTT) at a ﬂow-rate of 40 μl/min. Fractions of 50 μl were collected and analyzed by Western blot analysis. The proteins thyroglobulin (669 kDa), disclose their site of function. ferritin (440 kDa), catalase (232 kDa) and aldolase (158 kDa) from a A functional role of AP-3 at the level of the TGN is gel ﬁltration calibration kit (Pharmacia) were used as standards. supported by the observation that the binding of AP-3 to membranes is ARF1-dependent like that of the TGN- Production of brain cytosol depleted from AP-3 localized AP-1 (Simpson et al., 1997). Membrane proteins 100 μl of pig brain cytosol (5 mg/ml) were precleared with 20 μl S.aureus (Sigma) for 1 h at 4°C. Subsequently equal aliquots were that have been shown to interact with AP-1 include the incubated for 12 h with the antibody against the δ-subunit of AP-3, MPRs (Sosa et al., 1993; Mauxion et al., 1996, Glickman which was immobilized to S.aureus, or with S.aureus without antibody. et al., 1989), lamp-1 (Ho¨ning et al., 1996) and invariant After pelleting the S.aureus, the depletion step was repeated a second chain (Salamero et al., 1996). All these proteins are time. The ﬁnal supernatant was used for interaction analysis (see below). detected in early endosomes, and also to some extent at Peptide synthesis the plasma membrane. AP-3-mediated packaging of cargo All peptides used in this study were synthesized and puriﬁed as proteins at the TGN may, in contrast, initiate a pathway described (Ho¨ning et al., 1997). Their purity was conﬁrmed by HPLC, which bypasses the endosomal target of AP-1 dependent UV-spectrometry and mass-spectrometry. vesicles, resulting in a more direct delivery of proteins to SPR interaction analysis lysosomes (LIMP-II) or storage vesicles (tyrosinase). With The interaction between the different cytoplasmic tails and AP-1/AP-2 regard to this, the observation that LIMP-II is packed into or pig brain cytosolic fractions was analyzed in real-time by SPR coated vesicles at the TGN may be signiﬁcant (Barriocanal (Jonsson et al., 1991) using a BIAcore-2000 biosensor (Biacore AB). et al., 1986). An alternative model would allocate AP-3 The peptides were coupled to a CM5 sensor chip via their primary function to an endosomal compartment to function in the amino groups exactly according to the manufacturer’s instructions. 1312 LIMP-II tail and tyrosinase tail binding to AP3 Peptides that were available with an N-terminal cysteine residue were References coupled to the sensor chip using the thiol-coupling procedure. The Ahle,S., Mann,A., Eichelsbacher,U. and Ungewickell,E. (1988) Structural sensor chip surface is ﬁrst activated and derived to generate an active relationships between clathrin assembly proteins from the Golgi and disulﬁde on the chip surface. This group is then exchanged for the the plasma membrane. EMBO J., 7, 919–928. intrinsic thiol group of the peptide during peptide immobilization. Barriocanal,J., Bonifacino,J., Yuan,L. and Sandoval,I. (1986) Brieﬂy, the activation of the chip was carried out by the injection of Biosynthesis, glycosylation, movement through the golgi system, and 0.05 M N-hydroxysuccinimide/0.2 M N-ethyl-N’-dimethyl-aminopropyl- transport to lysosomes by an N-linked carbohydrate-independent carbodiimide for 4 min using a ﬂow-rate of 5 μl/min. The active mechanism of three lysosomal integral membrane proteins. J. Biol. disulﬁde was then introduced by a subsequent injection of 80 mM 2,2- Chem., 261, 16755–16763. pyridinyldithio-ethane-amine in 0.1 M borate buffer, pH 8.5, for 5 min. Beermann,F., Orlow,S.J., Boissy,R.E., Schmidt,A., Boissy,Y.L. and The immobilization was carried out by injecting the peptides at 0.1 mg/ml Lamoreux,M.L. (1995) Misrouting of tyrosinase with a truncated in 10 mM Na-acetate, pH 4.0, for 7 min. Excess reactive disulﬁdes and cytoplasmic tail as a result of the murine platinum [c(p)] mutation. non-covalently bound peptide were removed by a 5 min injection of 50 mM cysteine/1 M NaCl in 100 mM formate buffer, pH 4.3. Exp. Eye Res., 61, 599–607. All interaction experiments were performed with buffer A (see above) Boll,W., Gallusser,A. and Kirchhausen,T. (1995) Role of the regulatory at a ﬂow-rate of 20 μl/min unless otherwise stated. Association for 2 min domain of the EGF-receptor cytoplasmic tail in selective binding of was followed by dissociation for 2 min, during which buffer A was the clathrin-associated complex AP-2. Curr. Biol., 5, 1168–1178. perfused. A short pulse injection (15 s) of 20 mM NaOH/0.5% SDS Boll,W., Ohno,H., Zhou,S.Y., Rapoport,I., Cantley,L.C., Bonifacino,J.S. was used to regenerate the sensor chip surface after each experimental and Kirchhausen,T. (1996) Sequence requirements for the recognition cycle. The peptide-derived sensor chips remained stable and retained their of tyrosine-based endocytic signals by clathrin AP-2 complexes. speciﬁc binding capacity for 100 experimental cycles of association/ EMBO J., 15, 5789–5795. dissociation and regeneration. AP-1 and AP-2 were used at 100 nM; Cosson,P. and Letourneur,F. (1997) Coatomer (COPI)-coated vesicles: crude brain cytosol and the cytosolic fractions derived from gel ﬁltration role in intracellular transport and protein sorting. Curr. Opin. Cell (see above) were used at a ﬁnal protein concentration of ~50 μg/ml. Biol., 9, 484–487. Cowles,C., Odorizzi,G., Payne,G.S. and Emr,S.D. (1997a) The AP-3 Determination of kinetic rate constants complex is essential for cargo-selective transport to the yeast vacuole. The rate constants (k for association and k for dissociation) of the a d Cell, 91, 109–118. interaction between tail peptides and puriﬁed AP-1 or AP-2 were Cowles,C.R., Snyder,W.B., Burd,C.G. and Emr,S.D. (1997b) Novel calculated by using the evaluation software of the BIAcore 2000. The golgi to vacuole delivery pathway in yeast: identiﬁcation of a mathematical models that are used are described in more detail elsewhere sorting determinant and required transport component. EMBO J., 16, (O’Shannessy et al., 1993; Schuck and Minton 1996; Schuck, 1997). 2769–2782. Association was determined 15–20 s after switching from buffer ﬂow Dell’Angelica,E.C., Ooi,C.E. and Bonifacino,J.S. (1997a) Beta 3A- to adaptor solution to avoid distortions due to injection and mixing. The adaptin, a subunit of the adaptor-like complex AP-3. J. Biol. Chem., dissociation rate constants were determined 5–10 s after switching to 272, 15078–15084. buffer ﬂow. After a ﬁrst dissociation phase for ~30 s, further dissociation Dell’Angelica,E.C., Ohno,H., Ooi,C.E., Rabinovich,E., Roche,K.W. and of adaptors was very low. The association constant k , the dissociation Bonifacino,J.S. (1997b) AP-3: an adaptor-like protein complex with constant k and the calculation of the equilibrium constant K k /k d D d a ubiquitous expression. EMBO J., 16, 917–928. were determined by using the BIA evaluation software version 1.2, Delmarmol,V. and Beermann,F. (1996) Tyrosinase and related proteins assuming a ﬁrst order kinetic A B AB. The model used calculates in mammalian pigmentation. FEBS Lett., 381, 165–168. the association rate constant k and the steady state response level R a eq Denzer,K., Weber,B., Hillerehfeld,A., Vonﬁgura,K. and Pohlmann,R. by ﬁtting data to the equation: (1997) Identiﬁcation of three internalization sequences in the –(K C K )(t–t ) a n d 0 R R (1–e ) eq cytoplasmic tail of the 46 kDa mannose 6-phosphate receptor. Biochem. J., 326, 497–505. where t is the time in s; R , the steady state response level; and C, the eq Dietrich,J., Kastrup,J., Nielsen,B.L., Odum,N. and Geisler,C. (1997) molar concentration of adaptors in the injection solution. The steric Regulation and function of the CD3 gamma DxxxLL motif: a binding interference factor N, which describes the valency of the interaction site for adaptor protein-1 and adaptor protein-2 in vitro. J. Cell Biol., between the adaptors and the MPR46 tail, was set to 1. The dissociation 138, 271–281. rate constant k was determined by ﬁtting data to the equation: Glickman,J.A., Conibear,E. and Pearse,B.M.F. (1989) Speciﬁcity of –K (t–t ) d 0 R R e binding of clathrin adaptors to signals on the mannose-6-phosphate/ insulin-like growth factor II receptor. EMBO J., 8, 1041–1047. where R is the reponse level at the beginning time t of the dissociation 0 0 Gough,N. and Fambrough,D. (1997) Different steady state subcellular phase. This model, which has recently been applied to describe adaptor distributions of the three splice variants of lysosome-associated tail interaction (Heilker et al., 1996), is described in more detail elsewhere membrane protein lamp-2 are determined largely by the COOH- (Karlsson et al., 1991; O’Shannessy et al., 1993). It should be noted terminal amino acid residue. J. Cell Biol., 137, 1161–1169. that the models described above allow the determination of rate constants Heilker,R., Manning-Krieg,U., Zuber,J.F. and Spiess,M. (1996) In vitro without reaching equilibrium during the experimental cycle. The relative binding of clathrin adaptors to sorting signals correlates with rate constants given illustrate the afﬁnity differences between different tail peptides and are independent of adaptor concentration. endocytosis and basolateral sorting. EMBO J., 15, 2893–2899. Ho¨ning,S. and Hunziker,W. (1995) Cytoplasmic determinants involved in direct lysosomal sorting, endocytosis, and basolateral targetting of Electrophoresis and Western blotting The cytosolic fractions derived from gel ﬁltration were subjected to rat lgp120 (lamp-I) in MDCK cells. J. Cell Biol., 128, 321–332. SDS–PAGE analysis (Laemmli, 1970) and subsequently transferred onto Ho¨ning,S., Grifﬁth,J., Geuze,H.J. and Hunziker,W. (1996) The tyrosine- nitrocellulose membranes. The membranes were then probed for AP-2 based lysosomal targetting signal in lamp-1 mediates sorting into or AP-3 using the speciﬁc ﬁrst antibodies (see above) followed by golgi-derived clathrin-coated vesicles. EMBO J., 15, 5230–5239. horseradish peroxidase-labelled second antibodies, and detected by using Ho¨ning,S., Sosa,M., Hille-Rehfeld,A. and von Figura,K. (1997) The 46- the ECL system (Amersham). kDa mannose 6-phosphate receptor contains multiple binding sites for clathrin adaptors. J. Biol. Chem., 272, 19884–19890. Hunziker,W. and Geuze,H.J. (1996) Intracellular trafﬁcking of lysosomal Acknowledgements membrane proteins. BioEssays, 18, 379–389. Johnson,K.F. and Kornfeld,S. (1992) A His-Leu-Leu sequence near the We express our gratitude to Thomas C.Su¨dhof for the unlimited use of carboxyl terminus of the cytoplasmic domain of the cation-dependent his BIAcore and to Margaret Robinson for the gift of antibodies. The mannose 6-phosphate receptor is necessary for the lysosomal enzyme work of K.Neifer and B.Schmidt on peptide synthesis and puriﬁcation sorting function. J. Biol. Chem., 267, 17110–17115. is gratefully acknowledged. This work is supported by grants from Jonsson,U. et al. (1991) Real-time biospeciﬁc interaction analysis using the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen surface plasmon resonance and a sensor chip technology. Industrie to K.v.F., a grant of the Ministero de Ciencia y Cultura Espanol to I.V.S. and an EC grant (ERBFMAX-CT96-0058) to K.v.F. and I.V.S. Biotechniques, 11, 620–627. 1313 S.Ho¨ning, I.V.Sandoval and K.von Figura Karlsson,R., Michaelsson,A. and Mattsson,L. (1991) Kinetic analysis of tail, are critical for targetting of the resident lysosomal membrane protein monoclonal antibody–antigen interactions with a new biosensor based LIMP II to lysosomes. J. Biol. Chem., 269, 6622–6631. analytical system. J. Immun. Meth., 145, 229–240. Schraermeyer,U. (1995) Transport of endocytosed material into melanin Keen,J.H. (1987) Clathrin assembly proteins: afﬁnity puriﬁcation and a granules in cultured choroidal melanocytes of cattle—new insights into model for coat assembly. J. Cell Biol., 105, 1989–1998. the relationship of melanosomes with lysosomes. Pigm. Cell Res., 8, Keen,J.H., Willingham,M.C. and Pastan,I.H. (1979) Clathrin coated 209–214. vesicles: isolation, dissociation and factor-dependent reassociation of Schroder,S. and Ungewickell,E. (1991) Subunit interaction and function of clathrin-coated vesicle adaptors from the Golgi and the plasma clathrin baskets. Cell, 16, 303–312. membrane. J. Biol. Chem., 266, 7910–7918. Kirchhausen,T., Bonifacino,J.S. and Riezman,H. (1997) Linking cargo Schuck,P. (1997) Use of surface plasmon resonance to probe the to vesicle formation: receptor tail interactions with coat proteins. Curr. equilibrium and dynamic aspects of interactions between biological Opin. Cell Biol., 9, 488–495. macromolecules. Annu. Rev. Biophys. Biomol. Struc., 26, 541–566. Laemmli,U.K. (1970) Cleavage of structural proteins during assembly Schuck,P. and Minton,A.P. (1996) Analysis of mass transport-limited of the head of bacteriophage T4. Nature, 227, 680–685. binding kinetics in evanescent wave biosensors. Ann. Biochem., 240, Matter,K., Hunziker,W. and Mellman,I. (1992) Basolateral sorting of 262–272. LDL receptor in MDCK cells—the cytoplasmic domain contains 2 Simpson,F., Bright,N.A., West,M.A., Newman,L.S., Darnell,R.B. and Tyrosine-dependent targetting determinants. Cell, 71, 741–753. Robinson,M.S. (1996) A novel adaptor-related protein complex. J. Cell Matter,K., Yamamoto,E.M. and Mellman,I. (1994) Structural require- Biol., 133, 749–760. ments and sequence motifs for polarized sorting and endocytosis of Simpson,F., Peden,A.A., Christopoulou,L. and Robinson,M.S. (1997) LDL and fc receptors in MDCK cells. J. Cell Biol., 126, 991–1004. Characterization of the adaptor-related protein complex, AP-3. J. Cell Mauxion,F., LeBorgne,R., Munier-Lehmann,H. and Hoﬂack,B. (1996) Biol., 137, 835–845. A casein kinase II phosphorylation site in the cytoplasmic domain of Sosa,M.A., Schmidt,B., von Figura,K. and Hille-Rehfeld,A. (1993) In the cation-dependent mannose 6-phosphate receptor determines the vitro binding of plasma membrane-coated vesicle adaptors to the high afﬁnity interaction of the AP-1 golgi assembly proteins with cytoplasmic domain of lysosomal acid phosphatase. J. Biol. Chem., membranes. J. Biol. Chem., 271, 2171–2178. 268, 12537–12543. Newman,L.S., Mckeever,M.O., Okano,H.J. and Darnell,R.B. (1995) Stoorvogel,W., Oorschot,V. and Geuze,H.J. (1996) A novel class of Beta-NAP, a cerebellar degeneration antigen, is a neuron-speciﬁc clathrin-coated vesicles budding from endosomes. J. Cell Biol., 132, vesicle coat protein. Cell, 82, 773–783. 21–33. Ogata,S. and Fukuda,M. (1994) Lysosomal targetting of LIMP II Traub,L.M. (1997) Clathrin-associated adaptor proteins putting it all membrane glycoprotein requires a novel Leu-Ile motif at a particular together. Trends Cell Biol., 7, 43–46. position in its cytoplasmic tail. J. Biol. Chem., 269, 5210–5217. Vega,M., Segui-Real,B., Garcia,J., Cales,C., Rodriguez,F., Ohno,H. et al. (1995) Interaction of tyrosine-based sorting signals with Vanderkerckhove,J. and Sandoval,I. (1991) Cloning, sequencing, clathrin-associated proteins. Science, 269, 1872–1875. and expression of a cDNA encoding rat LIMP-II, a novel 74 kDa Ohno,H., Fournier,M.C., Poy,G. and Bonifacino,J.S. (1996) Structural lysosomal membrane protein related to the surface adhesion protein determinants of interaction of tyrosine-based sorting signals with the CD36. J. Biol. Chem., 266, 16818–16824. adaptor medium chains. J. Biol. Chem., 271, 29009–29015. Vijayasaradhi,S., Xu,Y.Q., Bouchard,B. and Houghton,A.N. (1995) Ooi,C.E., Moreira,J.E., Dell-Angelica,E.C., Poy,G., Wassarman,D.A. and Intracellular sorting and targetting of melanosomal membrane proteins: Bonifacino,J.S. (1997) Altered expression of a novel adaptin leads to identiﬁcation of signals for sorting of the human brown locus protein, defective pigment granule biogenesis in the drosophila eye color GP75. J. Cell Biol., 130, 807–820. mutant garnet. EMBO J., 16, 4508–4518. Wilde,A. and Brodsky,F.M. (1996) In vivo phosphorylation of adaptors Orlow,S.J. (1995) Melanosomes are specialized members of the regulates their interaction with clathrin. J. Cell Biol., 135, 635–645. lysosomal lineage of organelles. J. Invest. Dermatol., 105, 3–7. Orlow,S.J, Boissy,R.E., Moran,D.J. and Pifko-Hirst,S. (1993) Subcellular Received November 7, 1997; revised January 7, 1998; distribution of tyrosinase and tyrosinase-related protein-1: implications accepted January 8, 1998 for melanosomal biogenesis. J. Invest. Dermatol., 100, 55–64. O‘Shannessy,D., Brigham-Burk, M., Soneson,K., Hensley,P. and Brooks,I. (1993) Determination of rate and equilibrium constants for macromolecular interactions using surface plasmon resonance: Use of non linear least squares analysis methods. Ann. Biochem., 212, 457–468. Page,L.J. and Robinson,M.S. (1995) Targetting signals and subunit interactions in coated vesicle adaptor complexes. J. Cell Biol., 131, 619–630. Pearse,B.M.F. (1988) Receptors compete for adaptors found in plasma membrane coated pits. EMBO J., 7, 3331–3336. Pearse,B.M.F. and Robinson,M.S. (1990) Clathrin, adaptors and sorting. Annu. Rev. Cell Biol., 6, 151–171. Pond,L., Kuhn,L., Teyton,L., Schutze,M. and Peterson,P. (1995) A role for acidic residues in di-leucine motif-based targetting to the endocytosis pathway. J. Biol. Chem., 270, 19989–19997. Ponnambalam,S., Robinson,M.S., Jackson,A.P., Peiper,L. and Parham,P. (1990) Conservation and diversity in families of coated vesicles adaptins. J. Biol. Chem., 265, 4814–4820. Robinson,M.S. (1987) 100 kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies. J. Cell Biol., 104, 887–895. Robinson,M.S. (1997) Coats and vesicle budding. Trends Cell Biol., 7, 99–102. Salamero,J., LeBorgne,R., Saudrais,C., Goud,B. and Hoﬂack,B. (1996) Expression of major histocompatibility complex class II molecules in HeLa cells promotes the recruitment of AP-1 golgi-speciﬁc assembly proteins on golgi membranes. J. Biol. Chem., 271, 30318–30321. Sandoval,I.V. and Bakke,O. (1994) Targetting of membrane proteins to endosomes and lysosomes. Trends Cell Biol., 4, 292–297. Sandoval,I.V., Arredondo,J.J., Alcalde,J., Noriega,A.G., Vandekerckhov, J., Jimenez,M.A. and Rico,M. (1994) The residues Leu(Ile)(475)- Ile(Leu,Val,ALA)(476), contained in the extended carboxyl cytoplasmic

Journal

The EMBO Journal – Springer Journals

Published: Mar 2, 1998

Keywords: biosensor; coated vesicles; endosome; membrane traffic; protein sorting

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

A di‐leucine‐based motif in the cytoplasmic tail of LIMP‐II and tyrosinase mediates selective binding of AP‐3

A di‐leucine‐based motif in the cytoplasmic tail of LIMP‐II and tyrosinase mediates selective binding of AP‐3

Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

A di‐leucine‐based motif in the cytoplasmic tail of LIMP‐II and tyrosinase mediates selective binding of AP‐3

A di‐leucine‐based motif in the cytoplasmic tail of LIMP‐II and tyrosinase mediates selective binding of AP‐3

References (64)

Abstract

Journal

Recommended Articles

There are no references for this article.

Our policy towards the use of cookies