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Recognition of the polyubiquitin proteolytic signal

Recognition of the polyubiquitin proteolytic signal The EMBO Journal Vol.19 No.1 pp.94–102, 2000 with the ATP-dependent activation of Ub by an activating Julia S.Thrower, Laura Hoffman , 1 2 enzyme (E1). The ligation of ubiquitin to the substrate is Martin Rechsteiner and Cecile M.Pickart then carried out by a specific complex composed of a Department of Biochemistry and Molecular Biology, School of Public Ub–protein ligase (E3) and a Ub conjugating enzyme Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, (E2), with the E3 being the primary substrate specificity MD 21205 and Department of Biochemistry, School of Medicine, factor (Hershko and Ciechanover, 1998). During this University of Utah, Salt Lake City, UT 84132, USA recognition phase, many Ubs are ligated to the substrate, Corresponding author usually in the form of a polymeric chain (Chau et al., e-mail: [email protected] 1989). PolyUb chains linked through K48–G76 isopeptide bonds are the principal signal for proteasomal proteolysis Polyubiquitin chains linked through Lys48 are the (Chau et al., 1989; Finley et al., 1994). principal signal for targeting substrates to the 26S The 26S proteasome is a 2.1 MDa complex whose proteasome. Through studies of structurally defined, ~65 subunits are divided among three subcomplexes polyubiquitylated model substrates, we show that tetra- (Baumeister et al., 1998; Rechsteiner, 1998). One subcom- ubiquitin is the minimum signal for efficient protea- plex, the 20S proteasome, is a cylindrical stack of four somal targeting. The mechanism of targeting involves seven-membered rings. Its proteolytic active sites (six in a simple increase in substrate affinity that is brought eukaryotes) face an interior chamber that can be entered about by autonomous binding of the polyubiquitin only through a narrow pore at either end of the cylinder chain. Assigning the proteasomal signaling function to (Lo ¨we et al., 1995; Groll et al., 1997). Because folded a specific polymeric unit explains how a single ubiquitin proteins cannot reach this chamber, the isolated 20S can act as a functionally distinct signal, for example complex hydrolyzes only small peptides and denatured in endocytosis. The properties of the substrates studied proteins. The proteasome acquires activity toward folded here implicate substrate unfolding as a kinetically target proteins following the binding of one 19S complex dominant step in the proteolysis of properly folded to each end of the 20S cylinder. In general, a folded target proteins, and suggest that extraproteasomal chaperones protein is recognized by the 26S proteasome only if it has are required for efficient degradation of certain pro- been conjugated to a K48-linked polyUb chain (see Pickart, teasome substrates. 1997). The properties of the 26S proteasome suggest Keywords: chaperone/polyubiquitin/26S proteasome/ that the 19S complex mediates polyUb recognition and ubiquitin substrate unfolding. The use of a generalized signal, a polyUb chain, to target proteins for destruction is the defining characteristic Introduction of the Ub–proteasome pathway. If the 26S proteasome recognized its target proteins directly, then specificity Proteolysis is frequently used to regulate processes that would be restricted, as seen for the Clp and Lon proteases require rapid alterations in protein levels, including cell of Escherichia coli (Gottesman et al., 1997). Instead, cycle progression (e.g. Koepp et al., 1999). Most regu- target proteins are recognized by dedicated E2–E3 com- lated proteolysis in eukaryotes occurs by a mechanism in plexes. These enzymes generate the covalent polyUb which conjugation to the conserved protein ubiquitin (Ub) targeting signal, while the proteasome only needs to targets substrates for degradation by 26S proteasomes recognize this signal. The separation of target protein (Hochstrasser, 1996; Hershko and Ciechanover, 1998). recognition from the catalysis of peptide bond hydrolysis Substrates of the Ub–proteasome pathway include soluble is the key feature that allows the Ub–proteasome pathway proteins of the cytosol and nucleus, and proteins of the to degrade a remarkable array of substrates with high endoplasmic reticulum that have been ejected into the specificity. However, while several specific signals have cytoplasm (Sommer and Wolf, 1997). Ub also mediates been identified that lead to the assembly of polyUb chains the turnover of certain plasma membrane proteins by on substrate proteins (e.g. Koepp et al., 1999; Laney and targeting them for endocytosis, leading to proteolysis in Hochstrasser, 1999), little is yet known about polyUb the lysosome (Hicke, 1997). How the proteasomal and signal recognition and transduction. The major polyUb endocytic Ub targeting signals are distinguished is not yet receptor(s) in the 19S complex has not been identified, understood. the signal itself is incompletely characterized, and the Substrates of the Ub–proteasome pathway are marked molecular mechanism of targeting is poorly understood. for degradation by covalent ligation to Ub, which then acts as a signal for targeting the modified substrate to the We report an analysis of polyUb recognition by the proteasome. Ub is linked to the substrate through an proteasome that employed, for the first time, a structurally isopeptide bond between the C-terminus of Ub (G76) and defined polyubiquitylated substrate. The results reveal a lysine residue of the target protein. Ubiquitylation begins that tetraubiquitin constitutes the minimum proteasomal 94 © European Molecular Biology Organization The polyubiquitin proteolytic signal Fig. 2. Properties of Ub DHFR. All incubations except that in (D) contained Ubal. (A) Branched polyUb chain is essential for proteolytic targeting. Purified 26 proteasomes (~2 nM) were incubated with 35 35 150 nM of either [ S]Ub DHFR (circles) or [ S]UbDHFR (triangles) as described in Materials and methods. The rate of degradation of Fig. 1. Synthesis of Ub DHFR. (A) Scheme. UbDHFR has a polyHis Ub DHFR doubled when the proteasome concentration was doubled tag at its N-terminus and a hemagglutinin (HA) tag at its C-terminus. (not shown). (B) Dependence of initial degradation rate on substrate (B) Purification of [ S]UbDHFR and conjugation to Ub concentration. Results of two experiments are combined. Incubations (autoradiographs). Left, successive fractions in purification of contained 2.5 nM proteasomes; the curve is a least-squares fit of the 35 2 [ S]UbDHFR on Ni –NTA resin. Right, time course of Michaelis–Menten equation assuming K  35 nM. (C) Fused Ub [ S]UbDHFR conjugation to Ub . moiety of UbDHFR is degraded (Western blot). Proteasomes (~10 nM) were incubated with unlabeled Ub DHFR (75 nM). Aliquots were analyzed by blotting with antibodies against the polyHis tag of UbDHFR. The migration positions of Ub DHFR, UbDHFR and Ub targeting signal, explain the molecular basis of the depend- are indicated. Ub would migrate just above UbDHFR. Products ence of signal strength on chain length, and show that corresponding to the removal of one or two Ubs from the distal end of only a subset of potential interacting residues on the chain the polyUb chain of Ub DHFR are faintly visible in the second and third lanes, but represent only a small fraction of the starting substrate. surface is important for recognition. These findings suggest (D)Ub DHFR disassembly in the absence of Ubal (Western blot). that the higher-order conformation of the chain influences Proteasomes (~3 nM) were incubated with 100 nM Ub DHFR (see its signaling potential, and explain why a single Ub is an text). Aliquots were analyzed by blotting with antibodies against the inefficient proteasomal targeting signal. Unexpectedly, the C-terminal HA tag of Ub DHFR. Asterisks, deubiquitylated forms of substrates employed here, although recognized with high Ub DHFR. Note the different sampling times in (C) and (D). (E) Influence of polyUb chain length on proteolysis. Degradation of affinity, were slowly degraded. Several lines of evidence 35 35 [ S]Ub DHFR (triangles) or [ S]Ub DHFR (circles) was assayed in 5 9 suggest that this slow degradation reflects slow unfolding incubations with ~2 nM proteasomes. All rates were normalized to the of the target protein moiety. extrapolated V for Ub DHFR. The lines are fits of the Michaelis– max 5 Menten equation assuming K  68 nM (Ub DHFR, triangles) or M 5 K  14.5 nM (Ub DHFR, circles). The weaker binding of Ub DHFR M 9 5 Results relative to (B) reflects the use of different substrate and proteasome preparations. Model substrate for 26S proteasomes The ideal substrate for an in vitro analysis of proteasomal signal recognition should carry a homogeneous targeting active dihydrofolate reductase (Materials and methods), signal. The model substrate shown in Figure 1A features indicating that its DHFR moiety was properly folded a single Ub chain that is linked to one lysine residue (Stammers et al., 1987). The fused Ub moiety was also of the target protein. For the target protein we chose correctly folded, since it was recognized by E2-25K. dihydrofolate reductase (DHFR) fused at its N-terminus Ub DHFR was a well-behaved substrate for purified to Ub. UbDHFR acquires a polyUb chain and is targeted mammalian 26S proteasomes. Production of acid-soluble to proteasomes in yeast cells (Johnson et al., 1992, 1995). radioactivity from the labeled UbDHFR moiety of Although the UbDHFR conjugates seen in yeast feature a Ub DHFR was linear with time and depended on the K29 linkage in the polyUb chain (Johnson et al., 1995), presence of ATP (Figure 2A; data not shown). Degradation we reasoned that a homogeneous K48-linked chain would was also strictly dependent on the ligation of UbDHFR be sufficient to direct UbDHFR proteolysis, and this to Ub (Figure 2A) and was completely inhibited by proved to be correct. UbDHFR was metabolically labeled the well-characterized proteasome inhibitor MG-132 (not in E.coli and purified via an N-terminal polyHis tag shown; Rock et al., 1994). Ub DHFR was a high-affinity (Figure 1B). We then used the Ub-specific conjugating substrate (K  35 nM; Figure 2B). In a separate experi- enzyme E2-25K to link preassembled (K48-linked) Ub ment involving highly purified proteasomes, the molecular –1 to K48 in the Ub moiety of UbDHFR (Haldeman et al., turnover number (k ) was determined to be 0.05 min . cat 1997; Piotrowski et al., 1997; Figure 1). The final polyUb- Assuming that the ~380-residue UbDHFR protein is conjugated substrate, designated Ub DHFR, was a fully hydrolyzed to 10-residue peptides, k corresponds to 5 cat 95 J.S.Thrower et al. ~2 peptide bond cleavages/min/proteasome. The same pre- paration of 26S proteasomes hydrolyzed Suc-LLVY-AMC –1 with k  98 min , similar to previously reported values obs (e.g. Dick et al., 1991). The difference in the k values for cat a peptide versus Ub DHFR suggests that there is a slow step before peptide bond hydrolysis in the degradation of Ub DHFR (below). The Ub moiety of UbDHFR carries the G76V mutation to prevent its removal by deubiquitylating enzymes (Johnson et al., 1995). To test whether this fused Ub Fig. 3. Length dependence of unanchored polyUb chain binding to moiety was degraded, Ub DHFR was incubated with a 26S proteasomes. (A) Unanchored polyUb chains bind competitively with substrate. Incubations contained ~2 nM proteasomes, with 25, 45 high concentration of 26S proteasomes and reaction prod- or 67 nM [ S]Ub DHFR, and no Ub (circles), or 250 nM (triangles) 5 4 ucts were visualized by blotting with antibodies against or 500 nM (squares) Ub .(B) Inhibition versus chain length. the N-terminal polyHis tag of UbDHFR. If the fused Ub Incubations contained ~2 nM proteasomes, 100 nM [ S]Ub DHFR moiety escaped degradation, it would be converted to a and the indicated concentrations of unanchored Ub (circles), Ub 3 4 product of ~40 kDa (if linked to Ub ) or ~8 kDa (if released (squares) or Ub (triangles). Initial rates are expressed as a percentage 4 8 of the control reaction without unanchored chains. The curve is a from Ub ). Instead, most of the ~68-kDa Ub DHFR 4 5 least-squares fit of the equation v  (v K )/(K  [Ub ]) where 0 i 0.5 0.5 n protein disappeared without the production of smaller K  (1  [S]/K )K . For K values see Table I. 0.5 M i i immunoreactive products (Figure 2C). These results show that the fused Ub moiety is degraded. However, it may still be recognized as part of the polyUb chain (below). Table I. Length (n) and proximal end effects on polyUb chain binding to 26S proteasomes All degradation assays were carried out in the presence of Ub aldehyde (Ubal), a specific inhibitor of deubiquitylat- n Proximal end K (nM) K (Ub )/K (Ub ) i i n i 8 ing enzymes (Pickart and Rose, 1986; Hershko and Rose, 1987). When Ubal was omitted, the degradation of 2 Asp77 15 000 577 Ub DHFR was inhibited (not shown). Blotting with anti- 3 Asp77 1933  219 74 4 Asp77 171  16 6.6 body against a C-terminal epitope tag of Ub DHFR 6 Asp77 52  4 2.0 (Figure 2D) showed inhibition was due to disassembly of 8 Asp77 26  4 1.0 the substrate’s polyUb chain. Deubiquitylation, which was 12 Asp77 ~20 ~1.0 presumably due to the UCH37 subunit of the mammalian 4 diol 57  8 4 NAL 57  4 19S complex (Lam et al., 1997b), was efficiently sup- 5 βGal 35  4 pressed by Ubal (not shown), allowing us to monitor degradation exclusively. However, these findings suggest All values determined from inhibition of Ub DHFR degradation. Most that deubiquitylation and degradation could occur at com- values are the mean  SD, n  3. The value for Ub diol is from petitive rates on the proteasome in vivo (see Discussion). triplicate determinations at one chain concentration; the value for Ub βGal is from Figure 5. Ub is the minimum targeting signal We have suggested that the assembly of Ub into a K48- these hypotheses we studied the binding of different linked chain creates a unique recognition element that is length unanchored chains, as monitored by inhibition of bound by specific receptors in the 19S complex (Beal Ub DHFR degradation. We first established the validity et al., 1996, 1998). This model is consistent with the of unanchored chains as a model for substrate-linked chains defined conformation seen in the crystal structure of K48- by showing that inhibition by Ub could be overcome at linked Ub (Cook et al., 1994), and with the apparent a high concentration of Ub DHFR (Figure 3A). Such 4 5 inability of K63-linked chains to signal proteolysis in vivo competitive behavior indicates that the unanchored chain (Spence et al., 1995). However, it is also possible that the binds to the same site as the substrate. Studies with a assembly of Ub into a K48-linked chain enhances signaling series of unanchored chains revealed that affinity varied simply by increasing the concentration of monoUb with chain length (Figure 3B; data not shown). The (Pickart, 1997, 1998). These two models can be distingu- relationship appeared to be hyperbolic: the binding of Ub ished based on the length dependence of polyUb chain was too weak to be detected (K 15 μM), whereas Ub i 12 signaling. If the chain signals proteolysis by increasing and Ub bound with a similar high affinity (K ~25 nM; 8 i the concentration of Ub, then signaling should increase Table I). Because a 6-fold increase in chain length caused linearly with chain length; if the chain signals proteolysis an affinity increase of ~600-fold, the chain cannot signal by creating a new recognition element, then the depend- proteolysis by increasing the concentration of monoUb. ence is unlikely to be linear. Instead, Ub appears to be the minimum signal: affinity To investigate how signaling depends on chain length, increased ~100-fold as n increased from 2 to 4, but we first compared the substrate properties of UbDHFR 10-fold as n increased from 4 to 12 (Table I). conjugated to polyUb chains of different lengths. In The 6.6-fold difference in the affinities of Ub and Ub 8 4 comparison to Ub DHFR, Ub DHFR had a similar k (Table I) agrees well with the ~5-fold difference in the 5 9 cat and a 4.7-fold lower K (Figure 2E). These results K values of Ub DHFR and Ub DHFR (Figure 2E), M M 9 5 suggest that enhanced signaling is manifested as enhanced suggesting that the chain is the principal determinant of substrate affinity, and that affinity depends nonlinearly on substrate binding (below). The relative binding of Ub chain length (it will be shown below that K is equal to and Ub seen in Table I also agrees with the 6-fold M 4 the dissociation constant of the substrate). To confirm difference observed in a previous study (Piotrowski et al., 96 The polyubiquitin proteolytic signal 1997), but the K values determined earlier were app Table II. Binding of chimeric polyUb chains to 26S proteasomes ~200-fold weaker than the K values measured here. The earlier study employed a widely used proteasome substrate consisting of radiolabeled lysozyme conjugated to hetero- geneous length polyUb chains. It is likely that unlabeled conjugates, derived from target proteins contaminating the conjugating enzymes, were also present in this preparation (Piotrowski et al., 1997). The different results obtained in the two studies are consistent with this idea: the presence of such internal competitors would weaken the apparent binding of a given chain, but would not influence the relative binding of different length chains. The dissociation constant of unanchored Ub (170 nM) is 5-fold larger than the kinetic K of Ub DHFR (35 nM). M 5 This difference could reflect structural differences between the two chains: an unanchored chain has a negatively charged carboxylate at its proximal end, whereas a sub- strate-linked chain has a neutral isopeptide bond at this position. To test this hypothesis we conjugated Ub to N-acetyl lysine methyl ester, to make Ub NAL (Materials and methods). Ub NAL inhibited Ub DHFR degradation 4 5 with K  57 nM (Table I). The tighter binding of Ub NAL i 4 (in comparison with Ub ) is due to the absence of the negatively charged carboxylate, because converting the G76 carboxylate in Ub to an uncharged diol gave a similar increase in affinity (Table I). The affinities of Ub NAL and Ub diol are very similar to the K of 4 4 M Ub DHFR (57 versus 35 nM). The slightly higher affinity of Ub DHFR probably reflects a contribution of the fused Ub moiety to the recognition of the polyUb chain, i.e. it is as if DHFR is conjugated to Ub . Thus, the K 5 M value of Ub DHFR is essentially equal to its dissociation constant. These results show that Ub is a very efficient proteasomal targeting signal. Signal strength depends on the number of Ub Circles denote Ubs, numbered sequentially from the proximal end units (white, wild-type; dark, L8A). Reactions contained 0.13 μMUb The results shown in Table I suggest that Ub is the (chains 1–4) or 4 μMUb (chains 5–12), 150 nM [ S]Ub DHFR, and 4 5 minimum signal for efficient targeting to the proteasome, ~2 nM proteasomes. Inhibition of Ub DHFR degradation is expressed relative to a control without unanchored chains; values are but they do not explain why longer chains (n 4) bind means  SD (n 4). K is expressed relative to K of wt Ub (A) or i i 8 better than Ub . The latter result may be explained in two wt Ub (B). ways. In one model, longer chains bind better because they contain multiple Ub units. This model predicts that Ub will bind 3-fold more tightly than Ub , a factor which chimeric Ub molecules in which four recognition- 8 4 8 is similar to the observed ratios of 6.6-fold for unanchored deficient Ubs were incorporated at the proximal versus chains (Table I) and ~5-fold for chains conjugated to the distal end of the chain. As shown in Table IIA, the UbDHFR (Figure 2E). In a second model, only the incorporation of an L8A tetramer into Ub caused a proximal Ub unit is recognized, and chains of n 4 reduction in binding whose magnitude was independent bind better because lengthening the chain stabilizes the of the location of the mutant tetramer (compare chains 2 conformation of the proximal Ub unit. Our finding that and 3 with chain 1). Thus, the recognition of Ub does 4 4 the status of the chain’s proximal terminus influences not depend on its position within the chain. Moreover, binding (Table I) could be explained by this second model. each chimera bound to the proteasome with an affinity Both models predict that affinity will level off as the chain similar to that of Ub (~6-fold weaker binding than wild- becomes very long. They are distinguished by the location type Ub ; compare Table IIA with I), providing additional of the Ub unit responsible for targeting. In the first model, evidence that each Ub unit can be independently recog- 4 4 any Ub unit can be recognized, while in the second nized. These results show that Ub is the minimum 4 4 model only the proximal Ub unit is recognized for proteolytic signal, and that signal strength (affinity) productive binding. increases with the number of Ub units. Ub harboring the L8A mutation can be assembled into chains, but the mutant chains bind to the proteasome at Molecular features of the Ub signal least 15-fold more weakly than wild-type chains (Beal To define further the molecular properties of the Ub et al., 1996, 1998). Therefore, to differentiate between the targeting signal, we synthesized a series of chimeric Ub above-described models we compared the binding of molecules in which two L8A-Ubs were incorporated at 97 J.S.Thrower et al. various positions in the chain. In contrast to the data with the chimeric octamers, in which the position of a mutant tetramer did not alter binding (Table IIA), different chimeric tetramers had different affinities (Table IIB). The individual Ubs in Ub are therefore nonequivalent, as expected if Ub is the minimum targeting signal. In the nomenclature used here, the proximal Ub is defined to be the first Ub in the chain. Remarkably, placing L8A-Ub in the second and fourth positions had no effect on binding (compare chains 5 and 6), indicating either that these two Fig. 4. Ligands of DHFR inhibit degradation. (A) Tighter binding L8 residues do not interact with proteasomal receptors, or ligand is stronger inhibitor. Incubations contained ~2 nM proteasomes, that the energy derived from their interaction is used to 150 nM [ S]Ub DHFR and 100 μM of either folic acid (FA, triangles) or methotrexate (MTX, squares). (B) Noncompetitive drive an energetically unfavorable transition (see Mildvan inhibition by folic acid. Incubations contained ~2 nM proteasomes, et al., 1992). In contrast, the L8 side chains of the first with 25, 50 or 100 nM [ S]Ub DHFR, and 0 (squares), 20 (triangles) and third Ubs were clearly important for recognition or 40 (circles) μMFA. (compare chains 5 and 9). Comparison of the binding properties of chains 7–11 suggests that the L8 side chain of the first (proximal) Ub makes a stronger contribution competitive (not shown), suggesting that the linear chain to recognition than that of the third Ub. As in the case of occupies the site(s) occupied by the substrate’s K48-linked Ub (chain 4; Table I), the ability of an all-mutant chain chain. The properties of linear Ub provide the first direct 8 5 (chain 12) to bind, albeit with reduced affinity, suggests evidence that the linkage in a Ub polymer can influence that there are recognition determinants besides L8. The proteasomal signaling. Linear Ub is highly expressed in results also provide evidence of synergistic effects in the stressed cells, but it is co-translationally processed (Finley recognition of the elements of the Ub targeting signal. et al., 1987). Processing provides a high level of Ub for In the absence of such effects, changes in the free energy conjugation; our results suggest that it may also prevent of binding should be additive (Mildvan et al., 1992). inhibition of proteasomes. However, the sum of the change in free energy seen when mutating the first and third Ubs (0.8 kcal/mol, chain 9) Rate-limiting substrate unfolding and the change seen when mutating the second and fourth The polyUb chain of Ub DHFR is a potent targeting signal Ubs (0 kcal/mol, chain 6) underestimates the change due that fully accounts for this substrate’s interaction with to mutating all four Ubs (1.4 kcal/mol, chain 12). The proteasomes. However, despite its high affinity, Ub DHFR same conclusion follows from comparing chains 7, 10 is degraded ~50 times more slowly than a small peptide and 12. A more detailed knowledge of how chains bind (above). The most obvious difference between Ub DHFR to their cognate receptor(s) will be necessary before this and a peptide is that UbDHFR must be unfolded in order synergy can be interpreted at a mechanistic level. However, to be degraded. To test whether unfolding of UbDHFR the results summarized in Table IIB clearly indicate that limits the rate of degradation, we determined the effect the four Ubs in the Ub signal are non-equivalent. In of stabilizing this moiety through ligand binding. As contrast, there was no evidence for synergy in the recogni- shown in Figure 4A (squares), a saturating concentration tion of individual Ub units in Ub (Table IIA). of methotrexate (Appleman et al., 1988) almost completely 4 8 inhibited Ub DHFR degradation, as seen previously in Proteasomal binding of a linear polyUb chain reticulocyte lysate (Johnston et al., 1995). The same PolyUb chains linked through lysine residues other than concentration of folic acid (FA), which binds DHFR more K48 have been observed in vitro and in vivo (see Pickart, weakly (Mathews and Huennekens, 1983), inhibited the 1997, 1998). In particular, K63-linked chains have been degradation of Ub DHFR to a lesser extent (Figure 4A, implicated in processes that do not appear to depend on triangles). Inhibition by FA was noncompetitive targeting to the proteasome, including post-replicative (Figure 4B); the specific k effect indicates that the rate cat DNA repair (Spence et al., 1995; Hofmann and Pickart, of proteolysis of bound Ub DHFR decreases, as expected 1999) and endocytosis (Galan and Haguenauer-Tsapis, for rate-limiting unfolding. 1997). K63-linked chains could execute distinct signaling If k monitors unfolding, then its value should vary cat functions if they are conformationally distinct from K48- with substrate identity, because it is unlikely that two linked chains, resulting in differential binding to pro- different proteins will be unfolded at identical rates. To teasomes or other unidentified receptors. As a first test of test this prediction we studied the degradation of Ub βGal. this hypothesis, we characterized the proteasomal inter- Ub βGal was enzymatically active, indicating that it is a action of linear Ub . This chain is the product of the yeast tetramer of correctly folded 116 kDa subunits; each subunit UBI4 gene (Ozkaynak et al., 1987). Its constituent Ubs was conjugated to Ub (Jacobson et al., 1994; Materials are joined by G76-M1 peptide bonds. M1 is spatially and methods). Ub βGal was not detectably degraded by adjacent to K63 (Vijay-Kumar et al., 1987), suggesting 26S proteasomes (Figure 5A), but it bound tightly, as that linear Ub could resemble a K63-linked chain. Linear shown by its ability to inhibit Ub DHFR degradation 5 5 Ub inhibited Ub DHFR degradation with a reduced (Figure 5B). The K value of 35 nM (Figure 5B) shows 5 5 i affinity relative to K48-linked Ub (K  539 versus that the concentration of Ub βGal used in Figure 5A was 4 i 5 170 nM; data not shown). The true affinity difference is saturating. Therefore, k for Ub βGal is at least 50-fold cat 5 somewhat larger, because K48-linked Ub will bind more smaller than k for Ub DHFR. This difference is 5 cat 5 tightly than Ub (Table I). Inhibition by linear Ub was consistent with expectation for rate-limiting unfolding in 4 5 98 The polyubiquitin proteolytic signal Assigning the proteasomal signaling function to a specific polymeric unit allows a single Ub to act as a distinct type of signal, for example in endocytosis (Terrell et al., 1998). Ub is a remarkably efficient proteasomal targeting signal, cf. K ~60 nM for Ub conjugated to a blocked d 4 lysine residue. The affinity of Ub decreases when its proximal carboxylate is exposed (Table I). Although modest, this affinity difference will be advantageous in a cellular setting. Ub regeneration is thought to begin with release of the chain from a substrate remnant (Papa et al., Fig. 5. Substrate properties of Ub -βGal. (A) Inefficient degradation of 1999). This cleavage will facilitate dissociation of the Ub βGal. Incubations contained ~2 nM proteasomes and 200 nM 35 35 [ S]Ub DHFR (circles) or 400 nM [ S]Ub βGal (triangles). The unanchored chain from the proteasome (Table I); it will 5 5 concentration of Ub βGal refers to monomeric βGal subunits. 5 also strongly stimulate disassembly of the chain by the (B) Efficient binding of Ub βGal. Incubations contained ~2 nM enzyme known as isopeptidase T or Ubp14p (Wilkinson 35 35 proteasomes, 100 nM [ S]Ub DHFR, and 0 to 250 nM [ S]Ub βGal. 5 5 et al., 1995; Amerik et al., 1997). Together these effects The curve is a least-squares fit of the equation in Figure 3B, assuming will minimize inhibition of proteasomes by the polyUb K  35 nM. chain products of proteolysis. Our results convincingly demonstrate that a polyUb view of the greater structural complexity of βGal. Although chain is a universal targeting signal. The interaction of we could not detect degradation of Ub βGal by purified Ub DHFR with proteasomes is fully explained by the 5 5 26S proteasomes, polyUb-conjugated UbβGal is rapidly interaction of its attached polyUb chain, as shown by the degraded by proteasomes in yeast cells (Johnson et al., nearly identical affinities of Ub NAL and Ub DHFR and 4 5 1992, 1995; see Discussion). by the mutually exclusive binding of Ub and Ub DHFR. 4 5 The identical affinities of Ub βGal and Ub DHFR In addition, Ub DHFR and Ub βGal, substrates that feature 5 5 5 5 confirm that these substrates bind exclusively through identical polyUb chains but highly divergent target pro- their polyUb chains, and that Ub is a high-affinity teins, bind identically to proteasomes. For these properly targeting signal. However, our results provide no indication folded substrates the molecular mechanism of targeting that the chain performs any function besides targeting. It involves a very large increase in affinity that is brought has been suggested that the polyUb chain helps to unfold about by the autonomous binding of the polyUb chain. the substrate (Ghislain et al., 1996; see Pickart, 1997). We found no evidence that the chain affects downstream However, the unimpaired enzymatic activities of steps of proteasomal proteolysis. Nor did we observe trans- Ub DHFR and Ub βGal suggest that the chain does not targeting or allosteric effects: a saturating concentration of 5 5 destabilize the equilibrium folding of these target proteins. unanchored Ub did not make UbDHFR susceptible to A similar conclusion applies to polyubiquitylated forms proteasomal degradation or stimulate peptide hydrolysis of the plant photoreceptor phytochrome (Shanklin et al., by proteasomes (our unpublished data). Johnson et al. 1989). Nor was there evidence for an effect of the showed that the polyUb-conjugated subunit(s) of chimeric chain on the unfolding kinetics of the proteasome-bound βGal tetramers were selectively targeted for proteasomal substrate. Since unfolding is rate-limiting for degradation, degradation (Johnson et al., 1990). Similarly, proteasomes UbDHFR linked to polyUb chains of different lengths degrade ubiquitylated cyclins while sparing the associated should have been a sensitive reporter of such effects. cyclin-dependent kinase (Feldman et al., 1997; Skowyra Instead we found that k was independent of chain length et al., 1997; Koepp et al., 1999). The cis requirement in cat (Figure 2E). polyUb chain signaling is the key factor that allows the proteasome to remodel the compositions of such multisubunit complexes. Discussion An analysis of chimeric wild-type/L8A Ub molecules Function of the polyUb targeting signal shows that two of the four L8 residues in Ub contact Binding of the polyUb chain signal to its cognate recep- proteasomal receptors (Table IIB). In the crystal structure tor(s) in the 19S complex initiates the proteolysis of most of Ub each of these L8 residues is exposed on the same substrates of 26S proteasomes. An understanding of the face of Ub , while the other two L8 residues are exposed proteasome’s molecular mechanism must therefore begin on the opposite face (Cook et al., 1994; Beal et al., 1996). with an explication of polyUb recognition. Here we Our results can be explained if these two faces engage in used homogeneous K48-linked polyUb chains and novel distinct interactions when Ub is bound to its receptors in synthetic substrates to elucidate fundamental properties of the proteasome, suggesting that the conformation of Ub the polyUb proteolytic signal. Our results define Ub as is important for its recognition by proteasomal receptors. the minimum signal for efficient targeting. Ub is the Thus, different polyUb chains may act as distinct signals shortest chain that binds with high affinity to proteasomes in part because they have distinct conformations. The (K 1 μM), and n  4 defines a transition in the relation- proteasome-binding properties of linear Ub provide sup- d 5 ship between length and affinity (Table I). In addition, port for this model, but also suggest that this (artificial) any Ub unit in a chain can bind to proteasomes, in a chain retains significant proteasomal targeting potential. A manner that is independent of its location within the chain more rigorous determination of the proteasomal signaling (Table IIA). Finally, individual Ubs within Ub interact properties of an alternatively linked chain awaits the differently with proteasomal receptors (Table IIB), sug- availability of a substrate linked to such a chain. gesting that the Ub signal cannot be further subdivided. The results shown in Table II also place restrictions on 99 J.S.Thrower et al. the properties of authentic polyUb receptors in the 19S Cdc48p, also functions at a post-ubiquitylation step in the complex. A 50-kD protein of the mammalian 19S complex, degradation of IκBα (Dai et al., 1998). Cdc48p/VCP known as S5a, binds polyUb chains with high affinity thus represents one candidate for an extraproteasomal when assayed outside the complex (Deveraux et al., 1994). chaperone that could facilitate the degradation of folded, Although many of the polyUb binding properties of S5a polyubiquitylated substrates. However, although VCP is mimic those of proteasomes (Beal et al., 1996, 1998), no found associated with mammalian proteasomes (Dai et al., positional effects were seen in an analysis of the binding 1998), it is unlikely that it contributed significantly to the of chimeric wild-type/L8A Ub molecules to S5a (Beal binding or unfolding of Ub DHFR in our assays. This 4 5 et al., 1996). These results contrast with those shown in conclusion follows from our finding that k and K cat M Table IIB for proteasomes, a divergence that is consistent values for Ub DHFR were independent of substantial with the conclusion that S5a is not a major polyUb variations in the level of proteasome-associated VCP (our receptor of the proteasome (van Nocker et al., 1996; Fu unpublished data). A second way in which the proteolysis et al., 1998). of UbβGal (and UbDHFR) could be modulated is through changes in the structure of the polyUb chain. Data pre- Unfolding as a barrier to proteasomal degradation sented by Johnson et al. suggest that K29 in the fused Ub The rate-limiting step in the turnover of proteasome-bound moiety is the initial site of Ub ligation to UbDHFR, while Ub DHFR was assigned to unfolding based on the ability both K29 and K48 are utilized in UbβGal (Johnson et al., of DHFR ligands to decrease k , and on the precipitous 1995). The presence of K29 linkage(s) may recruit Ufd2p, cat decline in k that was seen upon replacing DHFR with a novel factor that modulates polyUb chain assembly cat the more complex βGal moiety. In addition, peptide (Koegl et al., 1999) and is required for UbDHFR proteo- hydrolysis by 26S proteasomes was unaffected by a lysis in yeast cells (Johnson et al., 1995). Ufd2p interacts saturating concentration of Ub DHFR (our unpublished with Cdc48p (Koegl et al., 1999), suggesting that there data), indicating that UbDHFR-derived material is essen- could even be a relationship between chain structure and tially absent from the hydrolytic active sites at Ub DHFR chaperone recruitment. saturation. The substrates used in our work featured a The efficient binding of Ub to proteasomes raises the fused Ub moiety that contributed the site for Ub conjuga- question of whether longer chains confer a significant tion. This moiety was engineered to resist removal by advantage in targeting. It is likely that the principal benefit deubiquitylating enzymes, and was degraded during the of lengthening the chain is not to increase the substrate’s proteolysis of Ub DHFR. Slow unfolding of the fused Ub affinity, but rather to increase its residence time on the moiety may contribute to the low k values of the synthetic proteasome (see Lam et al., 1997b). Once bound to the cat substrates. However, our data suggest that unfolding of mammalian 19S complex, chains are subject to the action the substrate moiety is also kinetically significant. This of the UCH37 deubiquitylating enzyme, which sequen- conclusion follows from the ability of specific DHFR tially removes Ubs from the distal chain terminus at a ligands to retard Ub DHFR degradation, and especially rate that is independent of chain length (Lam et al., from the finding that the two synthetic substrates had 1997a,b). If the substrate is conjugated to Ub , trimming different k values. the chain by just two Ubs will disrupt the minimal signal, cat We did not detect degradation of Ub βGal by purified causing a drop in affinity of ~100-fold. The substrate may 26S proteasomes. In marked contrast to this result, pulse– thus escape degradation. If the substrate is conjugated to chase measurements have yielded a half-life of 10 min Ub , trimming the chain by two Ubs will not change its for UbβGal in yeast cells (Johnson et al., 1992, 1995). affinity significantly. Our results suggest that the fate of This apparent discrepancy might be reconciled if a large a proteasome-bound substrate may be influenced by kinetic fraction of pulse-labeled Ub βGal molecules are degraded partitioning between deubiquitylation and unfolding. Such before they are completely folded. However, even folded partitioning could be made to favor destruction in at least βGal can be degraded in reticulocyte lysate with a half- two ways: by lengthening the chain so as to increase life as short as 1 h, despite having to undergo ubiquitylation the substrate’s residence time on the proteasome, or by in addition to proteasomal turnover (Gonda et al., 1989). recruiting a chaperone to increase the rate of substrate The properties of Ub βGal indicate that a polyUb chain unfolding. is not a universal degradation signal, even though it is a universal targeting signal. The slow turnover of this substrate by purified proteasomes can be explained in at Materials and methods least two ways. One possibility is that additional factors Plasmids and antibodies sometimes assist proteasomes in vivo. Given that unfolding pET3a-D77-Ub, pET3a-L8A,K48C-Ub and pET3a-L8A,D77-Ub were is a kinetically dominant step in turnover, these factors generated from pre-existing plasmids by standard procedures (Ausubel may include molecular chaperones. The 19S complex et al., 1995). pRS-5Ub-D77, encoding linear Ub with a 77th residue (Asp) in the final repeat, was from K.Wilkinson. pET16b-UbDHFR was harbors six subunits belonging to the AAA ATPase family V76 ΔK generated from pUb -V-e -DHFRha (Johnson et al., 1995) in two (Glickman et al., 1998; Rubin et al., 1998), but these steps. The complete insert was cloned into pET16b to introduce an N- subunits (and other intrinsic subunits of the 19S complex) ΔK terminal polyHis tag, and then the lacI-derived e domain was deleted are evidently unable to unfold the complex βGal molecule by ligating the small fragment of a BglII–EcoRI digest (encoding DHFRha) into the large fragment from a BamHI–EcoRI digest (encoding efficiently. Ghislain et al. showed that conditional mutation H -Ub). In the new fusion protein there is a four-residue linker (GSGI) of the yeast CDC48 gene, which encodes a chaperone of between Ub and mouse DHFR; Ub retains the G76V mutation which the AAA ATPase family, inhibits UbbGal turnover at a ΔK confers resistance to deubiquitylating enzymes. Deletion of the e post-ubiquitylation step (Ghislain et al., 1996). Valosin- domain enhanced the expression of UbDHFR and improved its solubility. containing protein (VCP), a mammalian homolog of Antibodies were from the following sources: Santa Cruz Biotechnology 100 The polyubiquitin proteolytic signal (anti-HA and anti-polyHis); Affiniti (anti-p45); and C.-C.H.Li (anti- preincubated for 8 min, initiated with proteasomes, and quenched by VCP). adding a reaction aliquot to a tube containing 2 vols of 10 mg/ml BSA, followed by 1 vol. of 40% (w/v) trichloroacetic acid. Degradation did Recombinant proteins not exceed 7% of input substrate in any reaction except that shown in Mutant Ubs were expressed and purified as described previously Figure 2C. Initial rates were usually determined from three time (Haldeman et al., 1997). Linear Ub was purified by subtractive anion points by least-squares linear regression analyses using Sigmaplot (see exchange followed by gradient cation exchange. [ S]H UbDHFR was Figure 2A). Similar results were obtained using different proteasome produced in E.coli strain BL21(DE3)pLysS at 37°C. Cells (100 ml) were preparations (types 1 and 2, above) and multiple preparations of grown to mid-log phase, washed twice with M9 medium, and resuspended Ub DHFR (k , K , and K values varied by no more than 2-fold). 5 cat M i in 50 ml of M9 medium containing 1% glucose, 0.063% methionine Hydrolysis of Suc-LLVY-AMC (0.1 mM, Bachem) was assayed at 37°C assay medium (Difco) and 2 mM FA. After 30 min, isopropyl-β-D- in incubations containing 50 mM Tris–HCl pH 7.6, 5 mM MgCl ,4mM galactopyranoside (IPTG) (0.4 mM) was added; after 30 min more, ATP, 10% glycerol and 1 mM DTT. rifampicin (150 μg/ml) was added. After 30 min more, [ S]methionine (2.5 mCi) was added for 5 min, followed by unlabeled methionine (1 mM) for 10 min more. Cells were harvested, frozen and lysed Acknowledgements (Haldeman et al., 1997). The clarified lysate was applied toa1ml Ni –NTA column and the fusion protein was purified by standard We thank R.Hofmann for assistance in several experiments, and the procedures, except that FA (2 mM) was included in all buffers, and following individuals for providing reagents: Y.Lam (purified pro- bovine serum albumin (BSA) was included as a carrier during elution. teasomes), C.-C.H.Li (anti-VCP antiserum), K.Wilkinson (pRS-5Ub) [ S]UbDHFR was exchanged into 5 mM HEPES pH 7.3, 0.1 mM and R.Cohen (Ubal, Ubdiol). For comments on the manuscript we thank EDTA, 1 mM dithiothreitol (DTT). Its concentration was estimated by R.Cohen, J.Bender and members of our laboratories. Funded by NIH SDS–PAGE (Coomassie Blue staining) using unlabeled UbDHFR as a grants DK46984 (C.P.) and GM37009 (M.R.), and by NCI training grant standard. [ S]UbβGal was expressed similarly, using pKK-UbGal in T32CA09110. JM101 cells (Gonda et al., 1989), and purified by affinity chromatography (Ullman, 1984). G76 of the Ub moiety in UbβGal is followed by Pro to inhibit the activity of deubiquitylating enzymes. References PolyUb chains Amerik,A.Y., Swaminathan,S., Krantz,B.A., Wilkinson,K.D. and K48-linked polyUb chains were assembled using E2-25K, which exclu- Hochstrasser,M. (1997) In vivo disassembly of free polyubiquitin sively recognizes K48 in Ub (Piotrowski et al., 1997 and references chains by yeast Ubp14 modulates rates of protein degradation by the therein). All chains carried the K48C mutation in the distal Ub; proteasome. EMBO J., 16, 4826–4838. unanchored chains carried D77 at the proximal terminus. These modifica- Appleman,J.R., Prendergast,N., Delcamp,T.J., Freisheim,J.H. and tions do not affect chain binding to 26S proteasomes (in comparison to Blakley,R.L. (1988) Kinetics of the formation and isomerization of chains carrying K48 and G76; Piotrowski et al., 1997). Ub diol was methotrexate complexes of recombinant human dihydrofolate made by using E2-25K to ligate Ub to Ubdiol (from R.Cohen; Lam reductase. J. Biol. Chem., 263, 10304–10313. et al., 1997a); in the case of Ub NAL, the substrates were Ub and NAL Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Smith,J.A. and 4 4 (0.1 M; Sigma). Struhl,K. (1995) Current Protocols in Molecular Biology. John Wiley and Sons, New York, NY. Proteasome substrates Baumeister,W., Walz,J., Zu ¨hl,F. and Seemu ¨ller,E. (1998) The proteasome: 35 4 [ S]UbDHFR (~4 μM, ~4  10 d.p.m./pmol) was incubated at 37°C paradigm of a self-compartmentalizing protease. Cell, 92, 367–380. overnight with Ub or Ub (90 μM), E1 (0.1 μM), C170S-E2-25K Beal,R., Deveraux,Q., Xia,G., Rechsteiner,M. and Pickart,C. (1996) 4 8 (15 μM) and yeast ubiquitin hydrolase-1 (YUH-1, 10 μg/ml) (Haldeman Surface hydrophobic residues of multiubiquitin chains essential for et al., 1997). (YUH-1 removes the proximal D77 residue, making Ub proteolytic targeting. Proc. Natl Acad. Sci. USA, 93, 861–866. competent to be conjugated by E2-25K.) FA (2 mM) was included to Beal,R.E., Toscano-Cantaffa,D., Young,P., Rechsteiner,M. and 35 35 stabilize UbDHFR. [ S]Ub DHFR and [ S]Ub DHFR were purified Pickart,C.M. (1998) The hydrophobic effect contributes to poly- 5 9 on Ni –NTA resin and concentrated into HEPES buffer (above), except ubiquitin chain recognition. Biochemistry, 37, 2925–2934. that all buffers contained 0.2 mg/ml BSA rather than FA. This step Chau,V., Tobias,J.W., Bachmair,A., Marriott,D., Ecker,D.J., Gonda,D.K. removed FA and unconjugated Ub .[ S]Ub βGal was synthesized and Varshavsky,A. (1989) A multiubiquitin chain is confined to specific 4 5 similarly and affinity purified. As in the synthesis of Ub DHFR, UbβGal lysine in a targeted short-lived protein. Science, 243, 1576–1583. was quantitatively converted to Ub βGal. Unlabeled Ub DHFR and Cook,W.J., Jeffrey,L.C., Kasperek,E. and Pickart,C.M. (1994) Structure 5 5 Ub βGal were synthesized similarly, except on a larger scale. The of tetraubiquitin shows how multiubiquitin chains can be formed. enzymatic activities of UbDHFR and UbβGal, and of their respective J. Mol. Biol., 236, 601–609. Ub conjugates, were measured as described (Mathews and Huennekens, Dai,R.-M., Chen,E., Longo,D.L., Gorbea,C.M. and Li,C.-C.H. (1998) 1983; Richard et al., 1985). Under standard conditions, UbDHFR and Involvement of valosin-containing protein, an ATPase co-purified Ub DHFR (38 nM) each consumed NADPH at a rate of 5.2 μmol/min, with IκBα and 26S proteasome, in ubiquitin–proteasome-mediated and identical βGal activities were seen for UbβGal versus Ub βGal. degradation of IκBα. J. Biol. Chem., 273, 3562–3573. Deveraux,Q., Ustrell,V., Pickart,C. and Rechsteiner,M. (1994) A 26S Proteasomes protease subunit that binds ubiquitin conjugates. J. Biol. Chem., 269, 26S proteasomes purified from bovine or rabbit erythrocytes, or rabbit 7059–7061. reticulocytes, were used interchangeably (Hoffman et al., 1992). Similar Dick,L.R., Moomaw,C.R., DeMartino,G.N. and Slaughter,C.A. (1991) results were obtained with proteasomes purified through the gradient Degradation of oxidized insulin B chain by the multiproteinase anion exchange step (preparation 1) versus proteasomes subjected to complex macropain (proteasome). Biochemistry, 30, 2725–2734. further purification on a glycerol gradient (preparation 2). The concentra- Dunten,R.L. and Cohen,R.E. (1989) Recognition of modified forms tion of proteasomes in preparation 2 was determined from the total of ribonuclease A by the ubiquitin system. J. Biol. Chem., 264, protein concentration assuming a molecular mass of 2.1 MDa. The 16739–16747. concentration of proteasomes in preparation 1 was estimated from Feldman,R.M.R., Correll,C.C., Kaplan,K.B. and Deshaies,R.J. (1997) A peptidase activity (based on the specific activity of preparation 2), and complex of Cdc4 , Skp1p and Cdc53p/cullin catalyzes ubiquitination confirmed by blotting with anti-p45/S8 antibodies. The two preparations of the phosphorylated CDK inhibitor Sic1p. Cell, 91, 221–230. had similar kinetic parameters in assays of Ub DHFR degradation. Finley,D., Ozkaynak,E. and Varshavsky,A. (1987) The yeast polyubiquitin gene is essential for resistance to high temperatures, Proteasome assays starvation and other stresses. Cell, 48, 1035–1046. Assays of the degradation of Ub DHFR and Ub βGal (25 ml, 37°C) Finley,D., Sadis,S., Monia,B.P., Boucher,P., Ecker,D.J., Crooke,S.T. and 5 5 contained: 50 mM Tris–HCl pH 7.6, 10 mM MgCl ,2 mM ATP, a Chau,V. (1994) Inhibition of proteolysis and cell cycle progression in phosphocreatine-based ATP-regenerating system, 10% glycerol, 0.4 mM a multiubiquitination-deficient yeast mutant. Mol. Cell. Biol., 14, DTT, 1 μM Ubal (except in Figure 2D) and 2 mg/ml BSA. Concentrations 5501–5509. of substrates and proteasomes are given in the legends to figures. Fu,H., Sadis,S., Rubin,D.M., Glickman,M., van Nocker,S., Finley,D. Ubal was from R.Cohen (Dunten and Cohen, 1989). Reactions were and Vierstra,R.D. (1998) Multiubiquitin chain binding and protein 101 J.S.Thrower et al. degradation are mediated by distinct domains within the 26S Papa,F.R., Amerik,A.Y. and Hochstrasser,M. (1999) Interaction of the proteasome subunit Mcb1. J. Biol. Chem., 273, 1970–1981. Doa4 deubiquitinating enzyme with the yeast 26S proteasome. Mol. Galan,J.-M. and Haguenauer-Tsapis,R. (1997) Ubiquitin Lys63 is Biol. Cell, 10, 741–756. involved in ubiquitination of a yeast plasma membrane protein. EMBO Pickart,C.M. (1997) Targeting of substrates to the 26S proteasome. J., 16, 5847–5854. FASEB J., 11, 1055–1066. Ghislain,M., Dohmen,R.J., Le ´vy,F. and Varshavsky,A. (1996) Cdc48p Pickart,C.M. (1998) Polyubiquitin chains. In Peters,J.-M., Harris,J.R. interacts with Ufd3p, a WD repeat protein required for ubiquitin- and Finley,D. (eds), Ubiquitin and the Biology of the Cell. Plenum mediated proteolysis in Saccharomyces cerevisiae. EMBO J., 15, Press, New York, NY, pp. 19–63. 4884–4899. Pickart,C.M. and Rose,I.A. (1986) Mechanism of ubiquitin carboxyl- Glickman,M.H., Rubin,D.M., Fried,V.A. and Finley,D. (1998) The terminal hydrolase: borohydride and hydroxylamine inactivate in regulatory particle of the Saccharomyces cerevisiae proteasome. Mol. presence of ubiquitin. J. Biol. Chem., 261, 10210–10217. Cell. Biol., 18, 3149–3162. Piotrowski,J., Beal,R., Hoffman,L., Wilkinson,K.D., Cohen,R.E. and Gonda,D.K., Bachmair,A., Wu ¨nning,I., Tobias,J.W., Lane,W.S. and Pickart,C.M. (1997) Inhibition of the 26S proteasome by polyubiquitin Varshavsky,A. (1989) Universality and structure of the N-end rule. J. chains synthesized to have defined lengths. J. Biol. Chem., 272, Biol. Chem., 264, 16700–16712. 23712–23721. Gottesman,S., Wickner,S. and Maurizi,M.R. (1997) Protein quality Rechsteiner,M. (1998) The 26S proteasome. In Peters,J.-M., Harris,J.R. control: triage by chaperones and proteases. Genes Dev., 11, 815–823. and Finley,D. (eds), Ubiquitin and the Biology of the Cell. Plenum Groll,M., Ditzel,L., Lo ¨we,J., Stock,D., Bochtler,M., Bartunik,H.D. and Press, New York, NY, pp. 147–190. Huber,R. (1997) Structure of 20S proteasome from yeast at 2.4 Å Richard,J.P., Westerfeld,J.G., Lin,S. and Beard,J. (1985) Structure- resolution. Nature, 386, 463–471. reactivity relationships for β-galactosidase (Escherichia coli, lacZ). Haldeman,M.T., Xia,G., Kasperek,E.M. and Pickart,C.M. (1997) Biochemistry, 34, 11713–11724. Structure and function of ubiquitin conjugating enzyme E2-25K: the Rock,K.L., Gramm,C., Rothstein,L, Clark,K., Stein,R., Dick,L., tail is a core-dependent activity element. Biochemistry, 36, 10526– Hwang,D. and Goldberg,A.L. (1994) Inhibitors of the proteasome 10537. block degradation of most cell proteins and the generation of peptides Hershko,H. and Rose,I.A. (1987) Ubiquitin-aldehyde: a general inhibitor presented on MHC class I molecules. Cell, 78, 761–771. of ubiquitin-recycling processes. Proc. Natl Acad. Sci. USA, 84, Rubin,D.M., Glickman,M.H., Larsen,C.N., Dhruvakaumar,S. and 1829–1833. Finley,D. (1998) Active site mutants in the six regulatory particle Hershko,A. and Ciechanover,A. (1998) The ubiquitin system. Annu. Rev. ATPases reveal multiple roles for ATP in the proteasome. EMBO J., Biochem., 67, 425–480. 17, 4909–4919. Hicke,L. (1997) Ubiquitin-dependent internalization of down-regulation Shanklin,J., Jabben,M. and Vierstra,R.D. (1989) Partial purification and of plasma membrane proteins. FASEB J., 11, 1215–1226. peptide mapping of ubiquitin–phytochrome conjugates from oat. Proc. Hochstrasser,M. (1996) Ubiquitin-dependent protein degradation. Annu. Natl Acad. Sci. USA, 28, 6028–6034. Rev. Genet., 30, 405–439. Skowyra,D., Craig,K.L., Tyers,M., Elledge,S.J. and Harper,J.W. (1997) Hoffman,L., Pratt,G. and Rechsteiner,M. (1992) Multiple forms of the F-box proteins are receptors that recruit phosphorylated substrates to 20S multicatalytic and the 26S ubiquitin/ATP-dependent proteases the SCF ubiquitin–ligase complex. Cell, 91, 209–219. from rabbit reticulocyte lysate. J. Biol. Chem., 267, 22362–22368. Sommer,T. and Wolf,D.H. (1997) Endoplasmic reticulum degradation: Hofmann,R.M. and Pickart,C.M. (1999) Noncanonical MMS2-encoded reverse protein flow of no return. FASEB J., 11, 1227–1233. ubiquitin conjugating enzyme functions in assembly of novel Spence,J., Sadis,S., Haas,A.L. and Finley,D. (1995) A ubiquitin mutant polyubiquitin chains for DNA repair. Cell, 96, 645–653. with specific defects in DNA repair and multiubiquitination. Mol. Jacobson,R.H., Zhang,X.J., DuBose,R.F. and Matthews,B.W. (1994) Cell. Biol., 15, 1265–1273. Three-dimensional structure of β-galactosidase from E.coli. Nature, Stammers,D.K., Champness,J.N., Beddell,C.R., Dann,J.G., 369, 761–766. Eliopoulous,E., Geddes,A.J., Ogg,D. and North,A.C. (1987) The Johnson,E.S., Gonda,D.K. and Varshavsky,A. (1990) Cis–trans structure of mouse L1210 dihydrofolate–drug complexes and the recognition and subunit specific degradation of short-lived proteins. construction of a model human enzyme. FEBS Lett., 218, 178–184. Nature, 346, 287–291. Terrell,J., Shih,S., Dunn,R. and Hicke,L. (1998) A function for Johnson,E.S., Bartel,B., Seufert,W. and Varshavsky,A. (1992) Ubiquitin monoubiquitination in the internalization of a G protein-coupled as a degradation signal. EMBO J., 11, 497–505. receptor. Mol. Cell, 1, 193–202. Johnson,E.S., Ma,P.C.M., Ota,I.M. and Varshavsky,A. (1995) A Ullman,A. (1984) One-step purification of hybrid proteins which have proteolytic pathway that recognizes ubiquitin as a degradation signal. β-galactosidase activity. Gene, 29, 27–31. J. Biol. Chem., 270, 17442–17456. van Nocker,S., Sadis,S., Rubin,D.M., Glickman,M., Fu,H., Coux,O., Johnston,J.A., Johnson,E.S., Waller,P.R. and Varshavsky,A. (1995) Wefes,I., Finley,D. and Vierstra,R.D. (1996) The multiubiquitin-chain- Methotrexate inhibits proteolysis of dihydrofolate reductase by the binding protein Mcb1 is a component of the 26S proteasome in N-end rule pathway. J. Biol. Chem., 270, 8172–8178. Saccharomyces cerevisiae and plays a nonessential, substrate-specific Koegl,M., Hoppe,T., Schlenker,S., Ulrich,H.D., Mayer,T.U. and role in protein turnover. Mol. Cell. Biol., 16, 6020–6028. Jentsch,S. (1999) A novel ubiquitination factor, E4, is involved in Vijay-Kumar,S., Bugg,C.E. and Cook,W.J. (1987) Structure of ubiquitin multiubiquitin chain assembly. Cell, 96, 635–644. refined to 1.8 Å resolution. J. Mol. Biol., 194, 531–544. Koepp,D.M., Harper,J.W. and Elledge,S.J. (1999) How the cyclin became Wilkinson,K.D., Tashayev,V.L., O’Connor,L.B., Larsen,C.N., a cyclin: regulated proteolysis in the cell cycle. Cell, 97, 431–434. Kasperek,E.M. and Pickart,C.M. (1995) Metabolism of the Lam,Y.A., DeMartino,G.N., Pickart,C.M. and Cohen,R.E. (1997a) polyubiquitin degradation signal: structure, mechanism and role of Specificity of the ubiquitin isopeptidase in the PA700 regulatory isopeptidase T. Biochemistry, 34, 14535–14546. complex of 26S proteasomes. J. Biol. Chem., 272, 28348–28446. Lam,Y.A., Xu.,W., DeMartino,G.N. and Cohen,R.E. (1997b) Editing of Received October 8, 1999; revised November 4, 1999; ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature, accepted November 10, 1999 385, 737–740. Laney,J.D. and Hochstrasser,M. (1999) Substrate targeting in the ubiquitin system. Cell, 97, 427–430. Lo ¨we,J., Stock,D., Jap,B., Zwickl,P., Baumeister,W. and Huber,R. (1995) Crystal structure of the 20S proteasome from the archaeon T.acidophilum at 3.4 Å resolution. Science, 268, 533–539. Mathews,C.K. and Huennekens,F.M. (1983) Further studies on dihydrofolic reductase. J. Biol. Chem., 238, 3436–3442. Mildvan,A.S., Weber,D.J. and Kuliopulos,A. (1992) Quantitative interpretations of double mutations of enzymes. Arch. Biochem. Biophys., 294, 327–340. Ozkaynak,E., Finley,D., Solomon,M.J. and Varshavsky,A. (1987) The yeast ubiquitin genes: a family of natural gene fusions. EMBO J., 6, 1429–1439. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Recognition of the polyubiquitin proteolytic signal

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Abstract

The EMBO Journal Vol.19 No.1 pp.94–102, 2000 with the ATP-dependent activation of Ub by an activating Julia S.Thrower, Laura Hoffman , 1 2 enzyme (E1). The ligation of ubiquitin to the substrate is Martin Rechsteiner and Cecile M.Pickart then carried out by a specific complex composed of a Department of Biochemistry and Molecular Biology, School of Public Ub–protein ligase (E3) and a Ub conjugating enzyme Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, (E2), with the E3 being the primary substrate specificity MD 21205 and Department of Biochemistry, School of Medicine, factor (Hershko and Ciechanover, 1998). During this University of Utah, Salt Lake City, UT 84132, USA recognition phase, many Ubs are ligated to the substrate, Corresponding author usually in the form of a polymeric chain (Chau et al., e-mail: [email protected] 1989). PolyUb chains linked through K48–G76 isopeptide bonds are the principal signal for proteasomal proteolysis Polyubiquitin chains linked through Lys48 are the (Chau et al., 1989; Finley et al., 1994). principal signal for targeting substrates to the 26S The 26S proteasome is a 2.1 MDa complex whose proteasome. Through studies of structurally defined, ~65 subunits are divided among three subcomplexes polyubiquitylated model substrates, we show that tetra- (Baumeister et al., 1998; Rechsteiner, 1998). One subcom- ubiquitin is the minimum signal for efficient protea- plex, the 20S proteasome, is a cylindrical stack of four somal targeting. The mechanism of targeting involves seven-membered rings. Its proteolytic active sites (six in a simple increase in substrate affinity that is brought eukaryotes) face an interior chamber that can be entered about by autonomous binding of the polyubiquitin only through a narrow pore at either end of the cylinder chain. Assigning the proteasomal signaling function to (Lo ¨we et al., 1995; Groll et al., 1997). Because folded a specific polymeric unit explains how a single ubiquitin proteins cannot reach this chamber, the isolated 20S can act as a functionally distinct signal, for example complex hydrolyzes only small peptides and denatured in endocytosis. The properties of the substrates studied proteins. The proteasome acquires activity toward folded here implicate substrate unfolding as a kinetically target proteins following the binding of one 19S complex dominant step in the proteolysis of properly folded to each end of the 20S cylinder. In general, a folded target proteins, and suggest that extraproteasomal chaperones protein is recognized by the 26S proteasome only if it has are required for efficient degradation of certain pro- been conjugated to a K48-linked polyUb chain (see Pickart, teasome substrates. 1997). The properties of the 26S proteasome suggest Keywords: chaperone/polyubiquitin/26S proteasome/ that the 19S complex mediates polyUb recognition and ubiquitin substrate unfolding. The use of a generalized signal, a polyUb chain, to target proteins for destruction is the defining characteristic Introduction of the Ub–proteasome pathway. If the 26S proteasome recognized its target proteins directly, then specificity Proteolysis is frequently used to regulate processes that would be restricted, as seen for the Clp and Lon proteases require rapid alterations in protein levels, including cell of Escherichia coli (Gottesman et al., 1997). Instead, cycle progression (e.g. Koepp et al., 1999). Most regu- target proteins are recognized by dedicated E2–E3 com- lated proteolysis in eukaryotes occurs by a mechanism in plexes. These enzymes generate the covalent polyUb which conjugation to the conserved protein ubiquitin (Ub) targeting signal, while the proteasome only needs to targets substrates for degradation by 26S proteasomes recognize this signal. The separation of target protein (Hochstrasser, 1996; Hershko and Ciechanover, 1998). recognition from the catalysis of peptide bond hydrolysis Substrates of the Ub–proteasome pathway include soluble is the key feature that allows the Ub–proteasome pathway proteins of the cytosol and nucleus, and proteins of the to degrade a remarkable array of substrates with high endoplasmic reticulum that have been ejected into the specificity. However, while several specific signals have cytoplasm (Sommer and Wolf, 1997). Ub also mediates been identified that lead to the assembly of polyUb chains the turnover of certain plasma membrane proteins by on substrate proteins (e.g. Koepp et al., 1999; Laney and targeting them for endocytosis, leading to proteolysis in Hochstrasser, 1999), little is yet known about polyUb the lysosome (Hicke, 1997). How the proteasomal and signal recognition and transduction. The major polyUb endocytic Ub targeting signals are distinguished is not yet receptor(s) in the 19S complex has not been identified, understood. the signal itself is incompletely characterized, and the Substrates of the Ub–proteasome pathway are marked molecular mechanism of targeting is poorly understood. for degradation by covalent ligation to Ub, which then acts as a signal for targeting the modified substrate to the We report an analysis of polyUb recognition by the proteasome. Ub is linked to the substrate through an proteasome that employed, for the first time, a structurally isopeptide bond between the C-terminus of Ub (G76) and defined polyubiquitylated substrate. The results reveal a lysine residue of the target protein. Ubiquitylation begins that tetraubiquitin constitutes the minimum proteasomal 94 © European Molecular Biology Organization The polyubiquitin proteolytic signal Fig. 2. Properties of Ub DHFR. All incubations except that in (D) contained Ubal. (A) Branched polyUb chain is essential for proteolytic targeting. Purified 26 proteasomes (~2 nM) were incubated with 35 35 150 nM of either [ S]Ub DHFR (circles) or [ S]UbDHFR (triangles) as described in Materials and methods. The rate of degradation of Fig. 1. Synthesis of Ub DHFR. (A) Scheme. UbDHFR has a polyHis Ub DHFR doubled when the proteasome concentration was doubled tag at its N-terminus and a hemagglutinin (HA) tag at its C-terminus. (not shown). (B) Dependence of initial degradation rate on substrate (B) Purification of [ S]UbDHFR and conjugation to Ub concentration. Results of two experiments are combined. Incubations (autoradiographs). Left, successive fractions in purification of contained 2.5 nM proteasomes; the curve is a least-squares fit of the 35 2 [ S]UbDHFR on Ni –NTA resin. Right, time course of Michaelis–Menten equation assuming K  35 nM. (C) Fused Ub [ S]UbDHFR conjugation to Ub . moiety of UbDHFR is degraded (Western blot). Proteasomes (~10 nM) were incubated with unlabeled Ub DHFR (75 nM). Aliquots were analyzed by blotting with antibodies against the polyHis tag of UbDHFR. The migration positions of Ub DHFR, UbDHFR and Ub targeting signal, explain the molecular basis of the depend- are indicated. Ub would migrate just above UbDHFR. Products ence of signal strength on chain length, and show that corresponding to the removal of one or two Ubs from the distal end of only a subset of potential interacting residues on the chain the polyUb chain of Ub DHFR are faintly visible in the second and third lanes, but represent only a small fraction of the starting substrate. surface is important for recognition. These findings suggest (D)Ub DHFR disassembly in the absence of Ubal (Western blot). that the higher-order conformation of the chain influences Proteasomes (~3 nM) were incubated with 100 nM Ub DHFR (see its signaling potential, and explain why a single Ub is an text). Aliquots were analyzed by blotting with antibodies against the inefficient proteasomal targeting signal. Unexpectedly, the C-terminal HA tag of Ub DHFR. Asterisks, deubiquitylated forms of substrates employed here, although recognized with high Ub DHFR. Note the different sampling times in (C) and (D). (E) Influence of polyUb chain length on proteolysis. Degradation of affinity, were slowly degraded. Several lines of evidence 35 35 [ S]Ub DHFR (triangles) or [ S]Ub DHFR (circles) was assayed in 5 9 suggest that this slow degradation reflects slow unfolding incubations with ~2 nM proteasomes. All rates were normalized to the of the target protein moiety. extrapolated V for Ub DHFR. The lines are fits of the Michaelis– max 5 Menten equation assuming K  68 nM (Ub DHFR, triangles) or M 5 K  14.5 nM (Ub DHFR, circles). The weaker binding of Ub DHFR M 9 5 Results relative to (B) reflects the use of different substrate and proteasome preparations. Model substrate for 26S proteasomes The ideal substrate for an in vitro analysis of proteasomal signal recognition should carry a homogeneous targeting active dihydrofolate reductase (Materials and methods), signal. The model substrate shown in Figure 1A features indicating that its DHFR moiety was properly folded a single Ub chain that is linked to one lysine residue (Stammers et al., 1987). The fused Ub moiety was also of the target protein. For the target protein we chose correctly folded, since it was recognized by E2-25K. dihydrofolate reductase (DHFR) fused at its N-terminus Ub DHFR was a well-behaved substrate for purified to Ub. UbDHFR acquires a polyUb chain and is targeted mammalian 26S proteasomes. Production of acid-soluble to proteasomes in yeast cells (Johnson et al., 1992, 1995). radioactivity from the labeled UbDHFR moiety of Although the UbDHFR conjugates seen in yeast feature a Ub DHFR was linear with time and depended on the K29 linkage in the polyUb chain (Johnson et al., 1995), presence of ATP (Figure 2A; data not shown). Degradation we reasoned that a homogeneous K48-linked chain would was also strictly dependent on the ligation of UbDHFR be sufficient to direct UbDHFR proteolysis, and this to Ub (Figure 2A) and was completely inhibited by proved to be correct. UbDHFR was metabolically labeled the well-characterized proteasome inhibitor MG-132 (not in E.coli and purified via an N-terminal polyHis tag shown; Rock et al., 1994). Ub DHFR was a high-affinity (Figure 1B). We then used the Ub-specific conjugating substrate (K  35 nM; Figure 2B). In a separate experi- enzyme E2-25K to link preassembled (K48-linked) Ub ment involving highly purified proteasomes, the molecular –1 to K48 in the Ub moiety of UbDHFR (Haldeman et al., turnover number (k ) was determined to be 0.05 min . cat 1997; Piotrowski et al., 1997; Figure 1). The final polyUb- Assuming that the ~380-residue UbDHFR protein is conjugated substrate, designated Ub DHFR, was a fully hydrolyzed to 10-residue peptides, k corresponds to 5 cat 95 J.S.Thrower et al. ~2 peptide bond cleavages/min/proteasome. The same pre- paration of 26S proteasomes hydrolyzed Suc-LLVY-AMC –1 with k  98 min , similar to previously reported values obs (e.g. Dick et al., 1991). The difference in the k values for cat a peptide versus Ub DHFR suggests that there is a slow step before peptide bond hydrolysis in the degradation of Ub DHFR (below). The Ub moiety of UbDHFR carries the G76V mutation to prevent its removal by deubiquitylating enzymes (Johnson et al., 1995). To test whether this fused Ub Fig. 3. Length dependence of unanchored polyUb chain binding to moiety was degraded, Ub DHFR was incubated with a 26S proteasomes. (A) Unanchored polyUb chains bind competitively with substrate. Incubations contained ~2 nM proteasomes, with 25, 45 high concentration of 26S proteasomes and reaction prod- or 67 nM [ S]Ub DHFR, and no Ub (circles), or 250 nM (triangles) 5 4 ucts were visualized by blotting with antibodies against or 500 nM (squares) Ub .(B) Inhibition versus chain length. the N-terminal polyHis tag of UbDHFR. If the fused Ub Incubations contained ~2 nM proteasomes, 100 nM [ S]Ub DHFR moiety escaped degradation, it would be converted to a and the indicated concentrations of unanchored Ub (circles), Ub 3 4 product of ~40 kDa (if linked to Ub ) or ~8 kDa (if released (squares) or Ub (triangles). Initial rates are expressed as a percentage 4 8 of the control reaction without unanchored chains. The curve is a from Ub ). Instead, most of the ~68-kDa Ub DHFR 4 5 least-squares fit of the equation v  (v K )/(K  [Ub ]) where 0 i 0.5 0.5 n protein disappeared without the production of smaller K  (1  [S]/K )K . For K values see Table I. 0.5 M i i immunoreactive products (Figure 2C). These results show that the fused Ub moiety is degraded. However, it may still be recognized as part of the polyUb chain (below). Table I. Length (n) and proximal end effects on polyUb chain binding to 26S proteasomes All degradation assays were carried out in the presence of Ub aldehyde (Ubal), a specific inhibitor of deubiquitylat- n Proximal end K (nM) K (Ub )/K (Ub ) i i n i 8 ing enzymes (Pickart and Rose, 1986; Hershko and Rose, 1987). When Ubal was omitted, the degradation of 2 Asp77 15 000 577 Ub DHFR was inhibited (not shown). Blotting with anti- 3 Asp77 1933  219 74 4 Asp77 171  16 6.6 body against a C-terminal epitope tag of Ub DHFR 6 Asp77 52  4 2.0 (Figure 2D) showed inhibition was due to disassembly of 8 Asp77 26  4 1.0 the substrate’s polyUb chain. Deubiquitylation, which was 12 Asp77 ~20 ~1.0 presumably due to the UCH37 subunit of the mammalian 4 diol 57  8 4 NAL 57  4 19S complex (Lam et al., 1997b), was efficiently sup- 5 βGal 35  4 pressed by Ubal (not shown), allowing us to monitor degradation exclusively. However, these findings suggest All values determined from inhibition of Ub DHFR degradation. Most that deubiquitylation and degradation could occur at com- values are the mean  SD, n  3. The value for Ub diol is from petitive rates on the proteasome in vivo (see Discussion). triplicate determinations at one chain concentration; the value for Ub βGal is from Figure 5. Ub is the minimum targeting signal We have suggested that the assembly of Ub into a K48- these hypotheses we studied the binding of different linked chain creates a unique recognition element that is length unanchored chains, as monitored by inhibition of bound by specific receptors in the 19S complex (Beal Ub DHFR degradation. We first established the validity et al., 1996, 1998). This model is consistent with the of unanchored chains as a model for substrate-linked chains defined conformation seen in the crystal structure of K48- by showing that inhibition by Ub could be overcome at linked Ub (Cook et al., 1994), and with the apparent a high concentration of Ub DHFR (Figure 3A). Such 4 5 inability of K63-linked chains to signal proteolysis in vivo competitive behavior indicates that the unanchored chain (Spence et al., 1995). However, it is also possible that the binds to the same site as the substrate. Studies with a assembly of Ub into a K48-linked chain enhances signaling series of unanchored chains revealed that affinity varied simply by increasing the concentration of monoUb with chain length (Figure 3B; data not shown). The (Pickart, 1997, 1998). These two models can be distingu- relationship appeared to be hyperbolic: the binding of Ub ished based on the length dependence of polyUb chain was too weak to be detected (K 15 μM), whereas Ub i 12 signaling. If the chain signals proteolysis by increasing and Ub bound with a similar high affinity (K ~25 nM; 8 i the concentration of Ub, then signaling should increase Table I). Because a 6-fold increase in chain length caused linearly with chain length; if the chain signals proteolysis an affinity increase of ~600-fold, the chain cannot signal by creating a new recognition element, then the depend- proteolysis by increasing the concentration of monoUb. ence is unlikely to be linear. Instead, Ub appears to be the minimum signal: affinity To investigate how signaling depends on chain length, increased ~100-fold as n increased from 2 to 4, but we first compared the substrate properties of UbDHFR 10-fold as n increased from 4 to 12 (Table I). conjugated to polyUb chains of different lengths. In The 6.6-fold difference in the affinities of Ub and Ub 8 4 comparison to Ub DHFR, Ub DHFR had a similar k (Table I) agrees well with the ~5-fold difference in the 5 9 cat and a 4.7-fold lower K (Figure 2E). These results K values of Ub DHFR and Ub DHFR (Figure 2E), M M 9 5 suggest that enhanced signaling is manifested as enhanced suggesting that the chain is the principal determinant of substrate affinity, and that affinity depends nonlinearly on substrate binding (below). The relative binding of Ub chain length (it will be shown below that K is equal to and Ub seen in Table I also agrees with the 6-fold M 4 the dissociation constant of the substrate). To confirm difference observed in a previous study (Piotrowski et al., 96 The polyubiquitin proteolytic signal 1997), but the K values determined earlier were app Table II. Binding of chimeric polyUb chains to 26S proteasomes ~200-fold weaker than the K values measured here. The earlier study employed a widely used proteasome substrate consisting of radiolabeled lysozyme conjugated to hetero- geneous length polyUb chains. It is likely that unlabeled conjugates, derived from target proteins contaminating the conjugating enzymes, were also present in this preparation (Piotrowski et al., 1997). The different results obtained in the two studies are consistent with this idea: the presence of such internal competitors would weaken the apparent binding of a given chain, but would not influence the relative binding of different length chains. The dissociation constant of unanchored Ub (170 nM) is 5-fold larger than the kinetic K of Ub DHFR (35 nM). M 5 This difference could reflect structural differences between the two chains: an unanchored chain has a negatively charged carboxylate at its proximal end, whereas a sub- strate-linked chain has a neutral isopeptide bond at this position. To test this hypothesis we conjugated Ub to N-acetyl lysine methyl ester, to make Ub NAL (Materials and methods). Ub NAL inhibited Ub DHFR degradation 4 5 with K  57 nM (Table I). The tighter binding of Ub NAL i 4 (in comparison with Ub ) is due to the absence of the negatively charged carboxylate, because converting the G76 carboxylate in Ub to an uncharged diol gave a similar increase in affinity (Table I). The affinities of Ub NAL and Ub diol are very similar to the K of 4 4 M Ub DHFR (57 versus 35 nM). The slightly higher affinity of Ub DHFR probably reflects a contribution of the fused Ub moiety to the recognition of the polyUb chain, i.e. it is as if DHFR is conjugated to Ub . Thus, the K 5 M value of Ub DHFR is essentially equal to its dissociation constant. These results show that Ub is a very efficient proteasomal targeting signal. Signal strength depends on the number of Ub Circles denote Ubs, numbered sequentially from the proximal end units (white, wild-type; dark, L8A). Reactions contained 0.13 μMUb The results shown in Table I suggest that Ub is the (chains 1–4) or 4 μMUb (chains 5–12), 150 nM [ S]Ub DHFR, and 4 5 minimum signal for efficient targeting to the proteasome, ~2 nM proteasomes. Inhibition of Ub DHFR degradation is expressed relative to a control without unanchored chains; values are but they do not explain why longer chains (n 4) bind means  SD (n 4). K is expressed relative to K of wt Ub (A) or i i 8 better than Ub . The latter result may be explained in two wt Ub (B). ways. In one model, longer chains bind better because they contain multiple Ub units. This model predicts that Ub will bind 3-fold more tightly than Ub , a factor which chimeric Ub molecules in which four recognition- 8 4 8 is similar to the observed ratios of 6.6-fold for unanchored deficient Ubs were incorporated at the proximal versus chains (Table I) and ~5-fold for chains conjugated to the distal end of the chain. As shown in Table IIA, the UbDHFR (Figure 2E). In a second model, only the incorporation of an L8A tetramer into Ub caused a proximal Ub unit is recognized, and chains of n 4 reduction in binding whose magnitude was independent bind better because lengthening the chain stabilizes the of the location of the mutant tetramer (compare chains 2 conformation of the proximal Ub unit. Our finding that and 3 with chain 1). Thus, the recognition of Ub does 4 4 the status of the chain’s proximal terminus influences not depend on its position within the chain. Moreover, binding (Table I) could be explained by this second model. each chimera bound to the proteasome with an affinity Both models predict that affinity will level off as the chain similar to that of Ub (~6-fold weaker binding than wild- becomes very long. They are distinguished by the location type Ub ; compare Table IIA with I), providing additional of the Ub unit responsible for targeting. In the first model, evidence that each Ub unit can be independently recog- 4 4 any Ub unit can be recognized, while in the second nized. These results show that Ub is the minimum 4 4 model only the proximal Ub unit is recognized for proteolytic signal, and that signal strength (affinity) productive binding. increases with the number of Ub units. Ub harboring the L8A mutation can be assembled into chains, but the mutant chains bind to the proteasome at Molecular features of the Ub signal least 15-fold more weakly than wild-type chains (Beal To define further the molecular properties of the Ub et al., 1996, 1998). Therefore, to differentiate between the targeting signal, we synthesized a series of chimeric Ub above-described models we compared the binding of molecules in which two L8A-Ubs were incorporated at 97 J.S.Thrower et al. various positions in the chain. In contrast to the data with the chimeric octamers, in which the position of a mutant tetramer did not alter binding (Table IIA), different chimeric tetramers had different affinities (Table IIB). The individual Ubs in Ub are therefore nonequivalent, as expected if Ub is the minimum targeting signal. In the nomenclature used here, the proximal Ub is defined to be the first Ub in the chain. Remarkably, placing L8A-Ub in the second and fourth positions had no effect on binding (compare chains 5 and 6), indicating either that these two Fig. 4. Ligands of DHFR inhibit degradation. (A) Tighter binding L8 residues do not interact with proteasomal receptors, or ligand is stronger inhibitor. Incubations contained ~2 nM proteasomes, that the energy derived from their interaction is used to 150 nM [ S]Ub DHFR and 100 μM of either folic acid (FA, triangles) or methotrexate (MTX, squares). (B) Noncompetitive drive an energetically unfavorable transition (see Mildvan inhibition by folic acid. Incubations contained ~2 nM proteasomes, et al., 1992). In contrast, the L8 side chains of the first with 25, 50 or 100 nM [ S]Ub DHFR, and 0 (squares), 20 (triangles) and third Ubs were clearly important for recognition or 40 (circles) μMFA. (compare chains 5 and 9). Comparison of the binding properties of chains 7–11 suggests that the L8 side chain of the first (proximal) Ub makes a stronger contribution competitive (not shown), suggesting that the linear chain to recognition than that of the third Ub. As in the case of occupies the site(s) occupied by the substrate’s K48-linked Ub (chain 4; Table I), the ability of an all-mutant chain chain. The properties of linear Ub provide the first direct 8 5 (chain 12) to bind, albeit with reduced affinity, suggests evidence that the linkage in a Ub polymer can influence that there are recognition determinants besides L8. The proteasomal signaling. Linear Ub is highly expressed in results also provide evidence of synergistic effects in the stressed cells, but it is co-translationally processed (Finley recognition of the elements of the Ub targeting signal. et al., 1987). Processing provides a high level of Ub for In the absence of such effects, changes in the free energy conjugation; our results suggest that it may also prevent of binding should be additive (Mildvan et al., 1992). inhibition of proteasomes. However, the sum of the change in free energy seen when mutating the first and third Ubs (0.8 kcal/mol, chain 9) Rate-limiting substrate unfolding and the change seen when mutating the second and fourth The polyUb chain of Ub DHFR is a potent targeting signal Ubs (0 kcal/mol, chain 6) underestimates the change due that fully accounts for this substrate’s interaction with to mutating all four Ubs (1.4 kcal/mol, chain 12). The proteasomes. However, despite its high affinity, Ub DHFR same conclusion follows from comparing chains 7, 10 is degraded ~50 times more slowly than a small peptide and 12. A more detailed knowledge of how chains bind (above). The most obvious difference between Ub DHFR to their cognate receptor(s) will be necessary before this and a peptide is that UbDHFR must be unfolded in order synergy can be interpreted at a mechanistic level. However, to be degraded. To test whether unfolding of UbDHFR the results summarized in Table IIB clearly indicate that limits the rate of degradation, we determined the effect the four Ubs in the Ub signal are non-equivalent. In of stabilizing this moiety through ligand binding. As contrast, there was no evidence for synergy in the recogni- shown in Figure 4A (squares), a saturating concentration tion of individual Ub units in Ub (Table IIA). of methotrexate (Appleman et al., 1988) almost completely 4 8 inhibited Ub DHFR degradation, as seen previously in Proteasomal binding of a linear polyUb chain reticulocyte lysate (Johnston et al., 1995). The same PolyUb chains linked through lysine residues other than concentration of folic acid (FA), which binds DHFR more K48 have been observed in vitro and in vivo (see Pickart, weakly (Mathews and Huennekens, 1983), inhibited the 1997, 1998). In particular, K63-linked chains have been degradation of Ub DHFR to a lesser extent (Figure 4A, implicated in processes that do not appear to depend on triangles). Inhibition by FA was noncompetitive targeting to the proteasome, including post-replicative (Figure 4B); the specific k effect indicates that the rate cat DNA repair (Spence et al., 1995; Hofmann and Pickart, of proteolysis of bound Ub DHFR decreases, as expected 1999) and endocytosis (Galan and Haguenauer-Tsapis, for rate-limiting unfolding. 1997). K63-linked chains could execute distinct signaling If k monitors unfolding, then its value should vary cat functions if they are conformationally distinct from K48- with substrate identity, because it is unlikely that two linked chains, resulting in differential binding to pro- different proteins will be unfolded at identical rates. To teasomes or other unidentified receptors. As a first test of test this prediction we studied the degradation of Ub βGal. this hypothesis, we characterized the proteasomal inter- Ub βGal was enzymatically active, indicating that it is a action of linear Ub . This chain is the product of the yeast tetramer of correctly folded 116 kDa subunits; each subunit UBI4 gene (Ozkaynak et al., 1987). Its constituent Ubs was conjugated to Ub (Jacobson et al., 1994; Materials are joined by G76-M1 peptide bonds. M1 is spatially and methods). Ub βGal was not detectably degraded by adjacent to K63 (Vijay-Kumar et al., 1987), suggesting 26S proteasomes (Figure 5A), but it bound tightly, as that linear Ub could resemble a K63-linked chain. Linear shown by its ability to inhibit Ub DHFR degradation 5 5 Ub inhibited Ub DHFR degradation with a reduced (Figure 5B). The K value of 35 nM (Figure 5B) shows 5 5 i affinity relative to K48-linked Ub (K  539 versus that the concentration of Ub βGal used in Figure 5A was 4 i 5 170 nM; data not shown). The true affinity difference is saturating. Therefore, k for Ub βGal is at least 50-fold cat 5 somewhat larger, because K48-linked Ub will bind more smaller than k for Ub DHFR. This difference is 5 cat 5 tightly than Ub (Table I). Inhibition by linear Ub was consistent with expectation for rate-limiting unfolding in 4 5 98 The polyubiquitin proteolytic signal Assigning the proteasomal signaling function to a specific polymeric unit allows a single Ub to act as a distinct type of signal, for example in endocytosis (Terrell et al., 1998). Ub is a remarkably efficient proteasomal targeting signal, cf. K ~60 nM for Ub conjugated to a blocked d 4 lysine residue. The affinity of Ub decreases when its proximal carboxylate is exposed (Table I). Although modest, this affinity difference will be advantageous in a cellular setting. Ub regeneration is thought to begin with release of the chain from a substrate remnant (Papa et al., Fig. 5. Substrate properties of Ub -βGal. (A) Inefficient degradation of 1999). This cleavage will facilitate dissociation of the Ub βGal. Incubations contained ~2 nM proteasomes and 200 nM 35 35 [ S]Ub DHFR (circles) or 400 nM [ S]Ub βGal (triangles). The unanchored chain from the proteasome (Table I); it will 5 5 concentration of Ub βGal refers to monomeric βGal subunits. 5 also strongly stimulate disassembly of the chain by the (B) Efficient binding of Ub βGal. Incubations contained ~2 nM enzyme known as isopeptidase T or Ubp14p (Wilkinson 35 35 proteasomes, 100 nM [ S]Ub DHFR, and 0 to 250 nM [ S]Ub βGal. 5 5 et al., 1995; Amerik et al., 1997). Together these effects The curve is a least-squares fit of the equation in Figure 3B, assuming will minimize inhibition of proteasomes by the polyUb K  35 nM. chain products of proteolysis. Our results convincingly demonstrate that a polyUb view of the greater structural complexity of βGal. Although chain is a universal targeting signal. The interaction of we could not detect degradation of Ub βGal by purified Ub DHFR with proteasomes is fully explained by the 5 5 26S proteasomes, polyUb-conjugated UbβGal is rapidly interaction of its attached polyUb chain, as shown by the degraded by proteasomes in yeast cells (Johnson et al., nearly identical affinities of Ub NAL and Ub DHFR and 4 5 1992, 1995; see Discussion). by the mutually exclusive binding of Ub and Ub DHFR. 4 5 The identical affinities of Ub βGal and Ub DHFR In addition, Ub DHFR and Ub βGal, substrates that feature 5 5 5 5 confirm that these substrates bind exclusively through identical polyUb chains but highly divergent target pro- their polyUb chains, and that Ub is a high-affinity teins, bind identically to proteasomes. For these properly targeting signal. However, our results provide no indication folded substrates the molecular mechanism of targeting that the chain performs any function besides targeting. It involves a very large increase in affinity that is brought has been suggested that the polyUb chain helps to unfold about by the autonomous binding of the polyUb chain. the substrate (Ghislain et al., 1996; see Pickart, 1997). We found no evidence that the chain affects downstream However, the unimpaired enzymatic activities of steps of proteasomal proteolysis. Nor did we observe trans- Ub DHFR and Ub βGal suggest that the chain does not targeting or allosteric effects: a saturating concentration of 5 5 destabilize the equilibrium folding of these target proteins. unanchored Ub did not make UbDHFR susceptible to A similar conclusion applies to polyubiquitylated forms proteasomal degradation or stimulate peptide hydrolysis of the plant photoreceptor phytochrome (Shanklin et al., by proteasomes (our unpublished data). Johnson et al. 1989). Nor was there evidence for an effect of the showed that the polyUb-conjugated subunit(s) of chimeric chain on the unfolding kinetics of the proteasome-bound βGal tetramers were selectively targeted for proteasomal substrate. Since unfolding is rate-limiting for degradation, degradation (Johnson et al., 1990). Similarly, proteasomes UbDHFR linked to polyUb chains of different lengths degrade ubiquitylated cyclins while sparing the associated should have been a sensitive reporter of such effects. cyclin-dependent kinase (Feldman et al., 1997; Skowyra Instead we found that k was independent of chain length et al., 1997; Koepp et al., 1999). The cis requirement in cat (Figure 2E). polyUb chain signaling is the key factor that allows the proteasome to remodel the compositions of such multisubunit complexes. Discussion An analysis of chimeric wild-type/L8A Ub molecules Function of the polyUb targeting signal shows that two of the four L8 residues in Ub contact Binding of the polyUb chain signal to its cognate recep- proteasomal receptors (Table IIB). In the crystal structure tor(s) in the 19S complex initiates the proteolysis of most of Ub each of these L8 residues is exposed on the same substrates of 26S proteasomes. An understanding of the face of Ub , while the other two L8 residues are exposed proteasome’s molecular mechanism must therefore begin on the opposite face (Cook et al., 1994; Beal et al., 1996). with an explication of polyUb recognition. Here we Our results can be explained if these two faces engage in used homogeneous K48-linked polyUb chains and novel distinct interactions when Ub is bound to its receptors in synthetic substrates to elucidate fundamental properties of the proteasome, suggesting that the conformation of Ub the polyUb proteolytic signal. Our results define Ub as is important for its recognition by proteasomal receptors. the minimum signal for efficient targeting. Ub is the Thus, different polyUb chains may act as distinct signals shortest chain that binds with high affinity to proteasomes in part because they have distinct conformations. The (K 1 μM), and n  4 defines a transition in the relation- proteasome-binding properties of linear Ub provide sup- d 5 ship between length and affinity (Table I). In addition, port for this model, but also suggest that this (artificial) any Ub unit in a chain can bind to proteasomes, in a chain retains significant proteasomal targeting potential. A manner that is independent of its location within the chain more rigorous determination of the proteasomal signaling (Table IIA). Finally, individual Ubs within Ub interact properties of an alternatively linked chain awaits the differently with proteasomal receptors (Table IIB), sug- availability of a substrate linked to such a chain. gesting that the Ub signal cannot be further subdivided. The results shown in Table II also place restrictions on 99 J.S.Thrower et al. the properties of authentic polyUb receptors in the 19S Cdc48p, also functions at a post-ubiquitylation step in the complex. A 50-kD protein of the mammalian 19S complex, degradation of IκBα (Dai et al., 1998). Cdc48p/VCP known as S5a, binds polyUb chains with high affinity thus represents one candidate for an extraproteasomal when assayed outside the complex (Deveraux et al., 1994). chaperone that could facilitate the degradation of folded, Although many of the polyUb binding properties of S5a polyubiquitylated substrates. However, although VCP is mimic those of proteasomes (Beal et al., 1996, 1998), no found associated with mammalian proteasomes (Dai et al., positional effects were seen in an analysis of the binding 1998), it is unlikely that it contributed significantly to the of chimeric wild-type/L8A Ub molecules to S5a (Beal binding or unfolding of Ub DHFR in our assays. This 4 5 et al., 1996). These results contrast with those shown in conclusion follows from our finding that k and K cat M Table IIB for proteasomes, a divergence that is consistent values for Ub DHFR were independent of substantial with the conclusion that S5a is not a major polyUb variations in the level of proteasome-associated VCP (our receptor of the proteasome (van Nocker et al., 1996; Fu unpublished data). A second way in which the proteolysis et al., 1998). of UbβGal (and UbDHFR) could be modulated is through changes in the structure of the polyUb chain. Data pre- Unfolding as a barrier to proteasomal degradation sented by Johnson et al. suggest that K29 in the fused Ub The rate-limiting step in the turnover of proteasome-bound moiety is the initial site of Ub ligation to UbDHFR, while Ub DHFR was assigned to unfolding based on the ability both K29 and K48 are utilized in UbβGal (Johnson et al., of DHFR ligands to decrease k , and on the precipitous 1995). The presence of K29 linkage(s) may recruit Ufd2p, cat decline in k that was seen upon replacing DHFR with a novel factor that modulates polyUb chain assembly cat the more complex βGal moiety. In addition, peptide (Koegl et al., 1999) and is required for UbDHFR proteo- hydrolysis by 26S proteasomes was unaffected by a lysis in yeast cells (Johnson et al., 1995). Ufd2p interacts saturating concentration of Ub DHFR (our unpublished with Cdc48p (Koegl et al., 1999), suggesting that there data), indicating that UbDHFR-derived material is essen- could even be a relationship between chain structure and tially absent from the hydrolytic active sites at Ub DHFR chaperone recruitment. saturation. The substrates used in our work featured a The efficient binding of Ub to proteasomes raises the fused Ub moiety that contributed the site for Ub conjuga- question of whether longer chains confer a significant tion. This moiety was engineered to resist removal by advantage in targeting. It is likely that the principal benefit deubiquitylating enzymes, and was degraded during the of lengthening the chain is not to increase the substrate’s proteolysis of Ub DHFR. Slow unfolding of the fused Ub affinity, but rather to increase its residence time on the moiety may contribute to the low k values of the synthetic proteasome (see Lam et al., 1997b). Once bound to the cat substrates. However, our data suggest that unfolding of mammalian 19S complex, chains are subject to the action the substrate moiety is also kinetically significant. This of the UCH37 deubiquitylating enzyme, which sequen- conclusion follows from the ability of specific DHFR tially removes Ubs from the distal chain terminus at a ligands to retard Ub DHFR degradation, and especially rate that is independent of chain length (Lam et al., from the finding that the two synthetic substrates had 1997a,b). If the substrate is conjugated to Ub , trimming different k values. the chain by just two Ubs will disrupt the minimal signal, cat We did not detect degradation of Ub βGal by purified causing a drop in affinity of ~100-fold. The substrate may 26S proteasomes. In marked contrast to this result, pulse– thus escape degradation. If the substrate is conjugated to chase measurements have yielded a half-life of 10 min Ub , trimming the chain by two Ubs will not change its for UbβGal in yeast cells (Johnson et al., 1992, 1995). affinity significantly. Our results suggest that the fate of This apparent discrepancy might be reconciled if a large a proteasome-bound substrate may be influenced by kinetic fraction of pulse-labeled Ub βGal molecules are degraded partitioning between deubiquitylation and unfolding. Such before they are completely folded. However, even folded partitioning could be made to favor destruction in at least βGal can be degraded in reticulocyte lysate with a half- two ways: by lengthening the chain so as to increase life as short as 1 h, despite having to undergo ubiquitylation the substrate’s residence time on the proteasome, or by in addition to proteasomal turnover (Gonda et al., 1989). recruiting a chaperone to increase the rate of substrate The properties of Ub βGal indicate that a polyUb chain unfolding. is not a universal degradation signal, even though it is a universal targeting signal. The slow turnover of this substrate by purified proteasomes can be explained in at Materials and methods least two ways. One possibility is that additional factors Plasmids and antibodies sometimes assist proteasomes in vivo. Given that unfolding pET3a-D77-Ub, pET3a-L8A,K48C-Ub and pET3a-L8A,D77-Ub were is a kinetically dominant step in turnover, these factors generated from pre-existing plasmids by standard procedures (Ausubel may include molecular chaperones. The 19S complex et al., 1995). pRS-5Ub-D77, encoding linear Ub with a 77th residue (Asp) in the final repeat, was from K.Wilkinson. pET16b-UbDHFR was harbors six subunits belonging to the AAA ATPase family V76 ΔK generated from pUb -V-e -DHFRha (Johnson et al., 1995) in two (Glickman et al., 1998; Rubin et al., 1998), but these steps. The complete insert was cloned into pET16b to introduce an N- subunits (and other intrinsic subunits of the 19S complex) ΔK terminal polyHis tag, and then the lacI-derived e domain was deleted are evidently unable to unfold the complex βGal molecule by ligating the small fragment of a BglII–EcoRI digest (encoding DHFRha) into the large fragment from a BamHI–EcoRI digest (encoding efficiently. Ghislain et al. showed that conditional mutation H -Ub). In the new fusion protein there is a four-residue linker (GSGI) of the yeast CDC48 gene, which encodes a chaperone of between Ub and mouse DHFR; Ub retains the G76V mutation which the AAA ATPase family, inhibits UbbGal turnover at a ΔK confers resistance to deubiquitylating enzymes. Deletion of the e post-ubiquitylation step (Ghislain et al., 1996). Valosin- domain enhanced the expression of UbDHFR and improved its solubility. containing protein (VCP), a mammalian homolog of Antibodies were from the following sources: Santa Cruz Biotechnology 100 The polyubiquitin proteolytic signal (anti-HA and anti-polyHis); Affiniti (anti-p45); and C.-C.H.Li (anti- preincubated for 8 min, initiated with proteasomes, and quenched by VCP). adding a reaction aliquot to a tube containing 2 vols of 10 mg/ml BSA, followed by 1 vol. of 40% (w/v) trichloroacetic acid. Degradation did Recombinant proteins not exceed 7% of input substrate in any reaction except that shown in Mutant Ubs were expressed and purified as described previously Figure 2C. Initial rates were usually determined from three time (Haldeman et al., 1997). Linear Ub was purified by subtractive anion points by least-squares linear regression analyses using Sigmaplot (see exchange followed by gradient cation exchange. [ S]H UbDHFR was Figure 2A). Similar results were obtained using different proteasome produced in E.coli strain BL21(DE3)pLysS at 37°C. Cells (100 ml) were preparations (types 1 and 2, above) and multiple preparations of grown to mid-log phase, washed twice with M9 medium, and resuspended Ub DHFR (k , K , and K values varied by no more than 2-fold). 5 cat M i in 50 ml of M9 medium containing 1% glucose, 0.063% methionine Hydrolysis of Suc-LLVY-AMC (0.1 mM, Bachem) was assayed at 37°C assay medium (Difco) and 2 mM FA. After 30 min, isopropyl-β-D- in incubations containing 50 mM Tris–HCl pH 7.6, 5 mM MgCl ,4mM galactopyranoside (IPTG) (0.4 mM) was added; after 30 min more, ATP, 10% glycerol and 1 mM DTT. rifampicin (150 μg/ml) was added. After 30 min more, [ S]methionine (2.5 mCi) was added for 5 min, followed by unlabeled methionine (1 mM) for 10 min more. Cells were harvested, frozen and lysed Acknowledgements (Haldeman et al., 1997). The clarified lysate was applied toa1ml Ni –NTA column and the fusion protein was purified by standard We thank R.Hofmann for assistance in several experiments, and the procedures, except that FA (2 mM) was included in all buffers, and following individuals for providing reagents: Y.Lam (purified pro- bovine serum albumin (BSA) was included as a carrier during elution. teasomes), C.-C.H.Li (anti-VCP antiserum), K.Wilkinson (pRS-5Ub) [ S]UbDHFR was exchanged into 5 mM HEPES pH 7.3, 0.1 mM and R.Cohen (Ubal, Ubdiol). For comments on the manuscript we thank EDTA, 1 mM dithiothreitol (DTT). Its concentration was estimated by R.Cohen, J.Bender and members of our laboratories. Funded by NIH SDS–PAGE (Coomassie Blue staining) using unlabeled UbDHFR as a grants DK46984 (C.P.) and GM37009 (M.R.), and by NCI training grant standard. [ S]UbβGal was expressed similarly, using pKK-UbGal in T32CA09110. JM101 cells (Gonda et al., 1989), and purified by affinity chromatography (Ullman, 1984). G76 of the Ub moiety in UbβGal is followed by Pro to inhibit the activity of deubiquitylating enzymes. References PolyUb chains Amerik,A.Y., Swaminathan,S., Krantz,B.A., Wilkinson,K.D. and K48-linked polyUb chains were assembled using E2-25K, which exclu- Hochstrasser,M. (1997) In vivo disassembly of free polyubiquitin sively recognizes K48 in Ub (Piotrowski et al., 1997 and references chains by yeast Ubp14 modulates rates of protein degradation by the therein). All chains carried the K48C mutation in the distal Ub; proteasome. EMBO J., 16, 4826–4838. unanchored chains carried D77 at the proximal terminus. These modifica- Appleman,J.R., Prendergast,N., Delcamp,T.J., Freisheim,J.H. and tions do not affect chain binding to 26S proteasomes (in comparison to Blakley,R.L. (1988) Kinetics of the formation and isomerization of chains carrying K48 and G76; Piotrowski et al., 1997). Ub diol was methotrexate complexes of recombinant human dihydrofolate made by using E2-25K to ligate Ub to Ubdiol (from R.Cohen; Lam reductase. J. Biol. Chem., 263, 10304–10313. et al., 1997a); in the case of Ub NAL, the substrates were Ub and NAL Ausubel,F.M., Brent,R., Kingston,R.E., Moore,D.D., Smith,J.A. and 4 4 (0.1 M; Sigma). Struhl,K. (1995) Current Protocols in Molecular Biology. John Wiley and Sons, New York, NY. Proteasome substrates Baumeister,W., Walz,J., Zu ¨hl,F. and Seemu ¨ller,E. (1998) The proteasome: 35 4 [ S]UbDHFR (~4 μM, ~4  10 d.p.m./pmol) was incubated at 37°C paradigm of a self-compartmentalizing protease. Cell, 92, 367–380. overnight with Ub or Ub (90 μM), E1 (0.1 μM), C170S-E2-25K Beal,R., Deveraux,Q., Xia,G., Rechsteiner,M. and Pickart,C. (1996) 4 8 (15 μM) and yeast ubiquitin hydrolase-1 (YUH-1, 10 μg/ml) (Haldeman Surface hydrophobic residues of multiubiquitin chains essential for et al., 1997). (YUH-1 removes the proximal D77 residue, making Ub proteolytic targeting. Proc. Natl Acad. Sci. USA, 93, 861–866. competent to be conjugated by E2-25K.) FA (2 mM) was included to Beal,R.E., Toscano-Cantaffa,D., Young,P., Rechsteiner,M. and 35 35 stabilize UbDHFR. [ S]Ub DHFR and [ S]Ub DHFR were purified Pickart,C.M. (1998) The hydrophobic effect contributes to poly- 5 9 on Ni –NTA resin and concentrated into HEPES buffer (above), except ubiquitin chain recognition. Biochemistry, 37, 2925–2934. that all buffers contained 0.2 mg/ml BSA rather than FA. This step Chau,V., Tobias,J.W., Bachmair,A., Marriott,D., Ecker,D.J., Gonda,D.K. removed FA and unconjugated Ub .[ S]Ub βGal was synthesized and Varshavsky,A. (1989) A multiubiquitin chain is confined to specific 4 5 similarly and affinity purified. As in the synthesis of Ub DHFR, UbβGal lysine in a targeted short-lived protein. Science, 243, 1576–1583. was quantitatively converted to Ub βGal. Unlabeled Ub DHFR and Cook,W.J., Jeffrey,L.C., Kasperek,E. and Pickart,C.M. (1994) Structure 5 5 Ub βGal were synthesized similarly, except on a larger scale. The of tetraubiquitin shows how multiubiquitin chains can be formed. enzymatic activities of UbDHFR and UbβGal, and of their respective J. Mol. Biol., 236, 601–609. Ub conjugates, were measured as described (Mathews and Huennekens, Dai,R.-M., Chen,E., Longo,D.L., Gorbea,C.M. and Li,C.-C.H. (1998) 1983; Richard et al., 1985). Under standard conditions, UbDHFR and Involvement of valosin-containing protein, an ATPase co-purified Ub DHFR (38 nM) each consumed NADPH at a rate of 5.2 μmol/min, with IκBα and 26S proteasome, in ubiquitin–proteasome-mediated and identical βGal activities were seen for UbβGal versus Ub βGal. degradation of IκBα. J. Biol. Chem., 273, 3562–3573. Deveraux,Q., Ustrell,V., Pickart,C. and Rechsteiner,M. (1994) A 26S Proteasomes protease subunit that binds ubiquitin conjugates. J. Biol. Chem., 269, 26S proteasomes purified from bovine or rabbit erythrocytes, or rabbit 7059–7061. reticulocytes, were used interchangeably (Hoffman et al., 1992). Similar Dick,L.R., Moomaw,C.R., DeMartino,G.N. and Slaughter,C.A. (1991) results were obtained with proteasomes purified through the gradient Degradation of oxidized insulin B chain by the multiproteinase anion exchange step (preparation 1) versus proteasomes subjected to complex macropain (proteasome). Biochemistry, 30, 2725–2734. further purification on a glycerol gradient (preparation 2). The concentra- Dunten,R.L. and Cohen,R.E. (1989) Recognition of modified forms tion of proteasomes in preparation 2 was determined from the total of ribonuclease A by the ubiquitin system. J. Biol. Chem., 264, protein concentration assuming a molecular mass of 2.1 MDa. The 16739–16747. concentration of proteasomes in preparation 1 was estimated from Feldman,R.M.R., Correll,C.C., Kaplan,K.B. and Deshaies,R.J. (1997) A peptidase activity (based on the specific activity of preparation 2), and complex of Cdc4 , Skp1p and Cdc53p/cullin catalyzes ubiquitination confirmed by blotting with anti-p45/S8 antibodies. The two preparations of the phosphorylated CDK inhibitor Sic1p. Cell, 91, 221–230. had similar kinetic parameters in assays of Ub DHFR degradation. Finley,D., Ozkaynak,E. and Varshavsky,A. (1987) The yeast polyubiquitin gene is essential for resistance to high temperatures, Proteasome assays starvation and other stresses. Cell, 48, 1035–1046. Assays of the degradation of Ub DHFR and Ub βGal (25 ml, 37°C) Finley,D., Sadis,S., Monia,B.P., Boucher,P., Ecker,D.J., Crooke,S.T. and 5 5 contained: 50 mM Tris–HCl pH 7.6, 10 mM MgCl ,2 mM ATP, a Chau,V. (1994) Inhibition of proteolysis and cell cycle progression in phosphocreatine-based ATP-regenerating system, 10% glycerol, 0.4 mM a multiubiquitination-deficient yeast mutant. Mol. Cell. Biol., 14, DTT, 1 μM Ubal (except in Figure 2D) and 2 mg/ml BSA. Concentrations 5501–5509. of substrates and proteasomes are given in the legends to figures. Fu,H., Sadis,S., Rubin,D.M., Glickman,M., van Nocker,S., Finley,D. Ubal was from R.Cohen (Dunten and Cohen, 1989). Reactions were and Vierstra,R.D. (1998) Multiubiquitin chain binding and protein 101 J.S.Thrower et al. degradation are mediated by distinct domains within the 26S Papa,F.R., Amerik,A.Y. and Hochstrasser,M. (1999) Interaction of the proteasome subunit Mcb1. J. Biol. Chem., 273, 1970–1981. Doa4 deubiquitinating enzyme with the yeast 26S proteasome. Mol. Galan,J.-M. and Haguenauer-Tsapis,R. (1997) Ubiquitin Lys63 is Biol. Cell, 10, 741–756. involved in ubiquitination of a yeast plasma membrane protein. EMBO Pickart,C.M. (1997) Targeting of substrates to the 26S proteasome. J., 16, 5847–5854. FASEB J., 11, 1055–1066. Ghislain,M., Dohmen,R.J., Le ´vy,F. and Varshavsky,A. (1996) Cdc48p Pickart,C.M. (1998) Polyubiquitin chains. In Peters,J.-M., Harris,J.R. interacts with Ufd3p, a WD repeat protein required for ubiquitin- and Finley,D. (eds), Ubiquitin and the Biology of the Cell. Plenum mediated proteolysis in Saccharomyces cerevisiae. EMBO J., 15, Press, New York, NY, pp. 19–63. 4884–4899. Pickart,C.M. and Rose,I.A. (1986) Mechanism of ubiquitin carboxyl- Glickman,M.H., Rubin,D.M., Fried,V.A. and Finley,D. (1998) The terminal hydrolase: borohydride and hydroxylamine inactivate in regulatory particle of the Saccharomyces cerevisiae proteasome. Mol. presence of ubiquitin. J. Biol. Chem., 261, 10210–10217. Cell. Biol., 18, 3149–3162. Piotrowski,J., Beal,R., Hoffman,L., Wilkinson,K.D., Cohen,R.E. and Gonda,D.K., Bachmair,A., Wu ¨nning,I., Tobias,J.W., Lane,W.S. and Pickart,C.M. (1997) Inhibition of the 26S proteasome by polyubiquitin Varshavsky,A. (1989) Universality and structure of the N-end rule. J. chains synthesized to have defined lengths. J. Biol. Chem., 272, Biol. Chem., 264, 16700–16712. 23712–23721. Gottesman,S., Wickner,S. and Maurizi,M.R. (1997) Protein quality Rechsteiner,M. (1998) The 26S proteasome. In Peters,J.-M., Harris,J.R. control: triage by chaperones and proteases. Genes Dev., 11, 815–823. and Finley,D. (eds), Ubiquitin and the Biology of the Cell. Plenum Groll,M., Ditzel,L., Lo ¨we,J., Stock,D., Bochtler,M., Bartunik,H.D. and Press, New York, NY, pp. 147–190. Huber,R. (1997) Structure of 20S proteasome from yeast at 2.4 Å Richard,J.P., Westerfeld,J.G., Lin,S. and Beard,J. (1985) Structure- resolution. Nature, 386, 463–471. reactivity relationships for β-galactosidase (Escherichia coli, lacZ). Haldeman,M.T., Xia,G., Kasperek,E.M. and Pickart,C.M. (1997) Biochemistry, 34, 11713–11724. Structure and function of ubiquitin conjugating enzyme E2-25K: the Rock,K.L., Gramm,C., Rothstein,L, Clark,K., Stein,R., Dick,L., tail is a core-dependent activity element. Biochemistry, 36, 10526– Hwang,D. and Goldberg,A.L. (1994) Inhibitors of the proteasome 10537. block degradation of most cell proteins and the generation of peptides Hershko,H. and Rose,I.A. (1987) Ubiquitin-aldehyde: a general inhibitor presented on MHC class I molecules. Cell, 78, 761–771. of ubiquitin-recycling processes. Proc. Natl Acad. Sci. USA, 84, Rubin,D.M., Glickman,M.H., Larsen,C.N., Dhruvakaumar,S. and 1829–1833. Finley,D. (1998) Active site mutants in the six regulatory particle Hershko,A. and Ciechanover,A. (1998) The ubiquitin system. Annu. Rev. ATPases reveal multiple roles for ATP in the proteasome. EMBO J., Biochem., 67, 425–480. 17, 4909–4919. Hicke,L. (1997) Ubiquitin-dependent internalization of down-regulation Shanklin,J., Jabben,M. and Vierstra,R.D. (1989) Partial purification and of plasma membrane proteins. FASEB J., 11, 1215–1226. peptide mapping of ubiquitin–phytochrome conjugates from oat. Proc. Hochstrasser,M. (1996) Ubiquitin-dependent protein degradation. Annu. Natl Acad. Sci. USA, 28, 6028–6034. Rev. Genet., 30, 405–439. Skowyra,D., Craig,K.L., Tyers,M., Elledge,S.J. and Harper,J.W. (1997) Hoffman,L., Pratt,G. and Rechsteiner,M. (1992) Multiple forms of the F-box proteins are receptors that recruit phosphorylated substrates to 20S multicatalytic and the 26S ubiquitin/ATP-dependent proteases the SCF ubiquitin–ligase complex. Cell, 91, 209–219. from rabbit reticulocyte lysate. J. Biol. Chem., 267, 22362–22368. Sommer,T. and Wolf,D.H. (1997) Endoplasmic reticulum degradation: Hofmann,R.M. and Pickart,C.M. (1999) Noncanonical MMS2-encoded reverse protein flow of no return. FASEB J., 11, 1227–1233. ubiquitin conjugating enzyme functions in assembly of novel Spence,J., Sadis,S., Haas,A.L. and Finley,D. (1995) A ubiquitin mutant polyubiquitin chains for DNA repair. Cell, 96, 645–653. with specific defects in DNA repair and multiubiquitination. Mol. Jacobson,R.H., Zhang,X.J., DuBose,R.F. and Matthews,B.W. (1994) Cell. Biol., 15, 1265–1273. Three-dimensional structure of β-galactosidase from E.coli. Nature, Stammers,D.K., Champness,J.N., Beddell,C.R., Dann,J.G., 369, 761–766. Eliopoulous,E., Geddes,A.J., Ogg,D. and North,A.C. (1987) The Johnson,E.S., Gonda,D.K. and Varshavsky,A. (1990) Cis–trans structure of mouse L1210 dihydrofolate–drug complexes and the recognition and subunit specific degradation of short-lived proteins. construction of a model human enzyme. FEBS Lett., 218, 178–184. Nature, 346, 287–291. Terrell,J., Shih,S., Dunn,R. and Hicke,L. (1998) A function for Johnson,E.S., Bartel,B., Seufert,W. and Varshavsky,A. (1992) Ubiquitin monoubiquitination in the internalization of a G protein-coupled as a degradation signal. EMBO J., 11, 497–505. receptor. Mol. Cell, 1, 193–202. Johnson,E.S., Ma,P.C.M., Ota,I.M. and Varshavsky,A. (1995) A Ullman,A. (1984) One-step purification of hybrid proteins which have proteolytic pathway that recognizes ubiquitin as a degradation signal. β-galactosidase activity. Gene, 29, 27–31. J. Biol. Chem., 270, 17442–17456. van Nocker,S., Sadis,S., Rubin,D.M., Glickman,M., Fu,H., Coux,O., Johnston,J.A., Johnson,E.S., Waller,P.R. and Varshavsky,A. (1995) Wefes,I., Finley,D. and Vierstra,R.D. (1996) The multiubiquitin-chain- Methotrexate inhibits proteolysis of dihydrofolate reductase by the binding protein Mcb1 is a component of the 26S proteasome in N-end rule pathway. J. Biol. Chem., 270, 8172–8178. Saccharomyces cerevisiae and plays a nonessential, substrate-specific Koegl,M., Hoppe,T., Schlenker,S., Ulrich,H.D., Mayer,T.U. and role in protein turnover. Mol. Cell. Biol., 16, 6020–6028. Jentsch,S. (1999) A novel ubiquitination factor, E4, is involved in Vijay-Kumar,S., Bugg,C.E. and Cook,W.J. (1987) Structure of ubiquitin multiubiquitin chain assembly. Cell, 96, 635–644. refined to 1.8 Å resolution. J. Mol. Biol., 194, 531–544. Koepp,D.M., Harper,J.W. and Elledge,S.J. (1999) How the cyclin became Wilkinson,K.D., Tashayev,V.L., O’Connor,L.B., Larsen,C.N., a cyclin: regulated proteolysis in the cell cycle. Cell, 97, 431–434. Kasperek,E.M. and Pickart,C.M. (1995) Metabolism of the Lam,Y.A., DeMartino,G.N., Pickart,C.M. and Cohen,R.E. (1997a) polyubiquitin degradation signal: structure, mechanism and role of Specificity of the ubiquitin isopeptidase in the PA700 regulatory isopeptidase T. Biochemistry, 34, 14535–14546. complex of 26S proteasomes. J. Biol. Chem., 272, 28348–28446. Lam,Y.A., Xu.,W., DeMartino,G.N. and Cohen,R.E. (1997b) Editing of Received October 8, 1999; revised November 4, 1999; ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature, accepted November 10, 1999 385, 737–740. Laney,J.D. and Hochstrasser,M. (1999) Substrate targeting in the ubiquitin system. Cell, 97, 427–430. Lo ¨we,J., Stock,D., Jap,B., Zwickl,P., Baumeister,W. and Huber,R. (1995) Crystal structure of the 20S proteasome from the archaeon T.acidophilum at 3.4 Å resolution. Science, 268, 533–539. Mathews,C.K. and Huennekens,F.M. (1983) Further studies on dihydrofolic reductase. J. Biol. Chem., 238, 3436–3442. Mildvan,A.S., Weber,D.J. and Kuliopulos,A. (1992) Quantitative interpretations of double mutations of enzymes. Arch. Biochem. Biophys., 294, 327–340. Ozkaynak,E., Finley,D., Solomon,M.J. and Varshavsky,A. (1987) The yeast ubiquitin genes: a family of natural gene fusions. EMBO J., 6, 1429–1439.

Journal

The EMBO Journal – Springer Journals

Published: Jan 4, 2000

Keywords: chaperone; polyubiquitin; 26S proteasome; ubiquitin

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