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Dual mechanisms specify Doa4‐mediated deubiquitination at multivesicular bodies

Dual mechanisms specify Doa4‐mediated deubiquitination at multivesicular bodies The EMBO Journal (2007) 26, 2454–2464 & 2007 European Molecular Biology Organization All Rights Reserved 0261-4189/07 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies where it deubiquitinates integral membrane proteins ubiqui- Caleb Richter, Matthew West tinated on their cytosolic domains. Monoubiquitination (or, and Greg Odorizzi* in some cases, polyubiquitination with a short chain of 2–3 Department of Molecular, Cellular, and Developmental Biology, Ub subunits) targets membrane proteins into lumenal MVB University of Colorado, Boulder, CO, USA vesicles formed by invagination of the late-endosomal mem- brane (Dupre and Haguenauer-Tsapis, 2001; Katzmann et al, Doa4 is a ubiquitin-specific protease in Saccharomyces 2001; Reggiori and Pelham, 2001; Urbanowski and Piper, cerevisiae that deubiquitinates integral membrane pro- 2001). Lumenal MVB vesicles and their cargoes are ultimately teins sorted into the lumenal vesicles of late-endosomal delivered into the hydrolytic interior of the vacuole upon multivesicular bodies (MVBs). We show that the non- fusion of the limiting endosomal membrane with the vacuo- catalytic N terminus of Doa4 mediates its recruitment to lar membrane. Doa4 ensures that Ub is recovered from endosomes through its association with Bro1, which is one cargoes before their enclosure within lumenal vesicles of several highly conserved class E Vps proteins that (Dupre and Haguenauer-Tsapis, 2001; Katzmann et al, 2001; comprise the core MVB sorting machinery. In turn, Bro1 Losko et al, 2001). Recruitment of Doa4 to late endosomes directly stimulates deubiquitination by interacting with a involves Bro1, one of several highly conserved ‘class E’ Vps YPxL motif in the catalytic domain of Doa4. Mutations in proteins that comprise the core machinery required for MVB either Doa4 or Bro1 that disrupt catalytic activation of cargo sorting and lumenal vesicle formation (Luhtala and Doa4 impair deubiquitination and sorting of MVB cargo Odorizzi, 2004). Bro1-mediated recruitment of Doa4 occurs proteins and lead to the formation of lumenal MVB vesi- downstream of cargo recognition, which is executed by Ub- cles that are predominantly small compared with the binding class E Vps proteins of the endosomal sorting com- vesicles seen in wild-type cells. Thus, by recruiting Doa4 plexes required for transport (ESCRTs) (Hurley and Emr, to late endosomes and stimulating its catalytic activity, 2006). Bro1 fulfills a novel dual role in coordinating deubiquiti- In mammalian cells, ubiquitinated membrane proteins are nation in the MVB pathway. similarly recognized by orthologs of the class E Vps machin- The EMBO Journal (2007) 26, 2454–2464. doi:10.1038/ ery and deubiquitinated by UBPY, the mammalian ortholog of sj.emboj.7601692; Published online 19 April 2007 Doa4 (Mizuno et al, 2005; Row et al, 2006). Mammalian class Subject Categories: membranes & transport E Vps proteins are also commandeered by many enveloped Keywords: deubiquitination; endosomes; multivesicular viruses to facilitate budding of infectious virions from host bodies; protein sorting cells, which is a Ub-dependent process topologically equiva- lent to the budding of lumenal MVB vesicles (Morita and Sundquist, 2004). Class E Vps proteins are recruited to the site of viral budding through interaction with ‘late domain’ Introduction motifs in virally encoded proteins. Alix, the mammalian ortholog of Bro1, binds the YPxL late domain motif in the Deubiquitinating enzymes (DUBs) catalyze removal of ubi- Gag subunit of human immunodeficiency virus-1 (HIV-1), quitin (Ub) from proteins targeted for degradation, thereby equine infectious anemia virus (EIAV), and murine leukemia replenishing the pool of free cellular Ub. DUBs also function virus (MuLV) (Martin-Serrano et al, 2003; Strack et al, 2003; in non-proteolytic processes such as cleavage of inactive Segura-Morales et al, 2005). Transplantation of YPxL into a poly-Ub translational fusions and deubiquitination of his- recombinant retrovirus results in enhanced deubiquitination tones, which regulate chromosome condensation and tran- of Gag (Martin-Serrano et al, 2004), suggesting that Alix scriptional silencing during mitosis (Kim et al, 2003; Amerik mediates recruitment of UBPY (or a different DUB) in a and Hochstrasser, 2004). The Ub-specific protease (UBP) manner analogous to recruitment of Doa4 by Bro1. family encompasses the majority of DUBs identified by In addition to its role in the MVB pathway, Doa4 functions sequence homology (Clague and Urbe, 2006). Many UBPs in removal of poly-Ub from soluble proteins targeted for are coupled to specific cellular processes, but the factors that degradation at proteasomes (Papa et al, 1999) and is impli- determine their functional specificities are largely uncharac- cated in the control of DNA replication and repair (Singer terized. et al, 1996; Fiorani et al, 2004). Regulating Doa4 specificity in Doa4 is a UBP in Saccharomyces cerevisiae that functions in multiple cellular processes is likely to be dependent upon the multivesicular body (MVB) protein sorting pathway, mechanisms that control its subcellular localization. However, we report that, alone, localization of Doa4 to *Corresponding author. Department of Molecular, Cellular, and Developmental Biology, University of Colorado, 347 UCB, Colorado endosomes is insufficient for specifying deubiquitination of Avenue, Boulder, CO 80309, USA. Tel.: þ 1 303 735 0179; MVB cargo proteins. Doa4 must also be activated upon its Fax: þ 1 303 492 7744; E-mail: [email protected] recruitment to endosomal membranes. Bro1 fulfills both roles by associating with the non-catalytic N-terminal region Received: 29 November 2006; accepted: 29 March 2007; published online: 19 April 2007 of Doa4 to mediate its localization to endosomes and by 2454 The EMBO Journal VOL 26 NO 10 2007 &2007 European Molecular Biology Organization | | Doa4-mediated deubiquitination at endosomes C Richter et al interacting with the catalytic domain of Doa4 to stimulate deubiquitination. Enzymatic activation of Doa4 occurs through binding of a conserved proline-based sequence located near the C terminus of Bro1 to a YPxL motif in Doa4. Mutations that prevent Bro1 from activating Doa4 disrupt deubiquitination and sorting of MVB cargo proteins. Thus, Bro1 coordinates Doa4 function and specificity by recruiting Doa4 to the MVB and acting as a cofactor to enhance deubiquitination. Results Bro1 associates with the N-terminal region of Doa4 to mediate its recruitment to endosomes Doa4 is one of 16 UBPs in S. cerevisiae, all of which contain highly conserved C-terminal catalytic domains coupled to N-terminal regions with low similarity among one another (Amerik and Hochstrasser, 2004). The N-terminal regions of UBPs are thought to determine substrate specificity, poten- tially by mediating localization of each UBP to a distinct subcellular site of function (Kim et al, 2003). Indeed, GFP fused to amino acids 1–560 of Doa4 (Doa4 -GFP; Figure 1A) localized to FM4-64-stained endosomes as efficiently as did GFP fused to full-length Doa4 (Doa4-GFP; Figure 1B). Both Doa4 -GFP and Doa4-GFP were observed in cells in which the VPS4 gene had been deleted (vps4D). VPS4 encodes an ATPase that catalyzes dissociation of Doa4 (and class E Vps proteins) from endosomal membranes. Thus, the absence of Vps4 facilitates identification of Doa4 recruitment by trapping it at endosomes (Luhtala and Odorizzi, 2004). Unlike Doa4 -GFP and Doa4-GFP, GFP was predominantly cytosolic in vps4D cells when fused to amino acids 561–926 of CAT Doa4, which comprise its catalytic domain (Doa4 -GFP; Figure 1C). The N terminus of Doa4, therefore, is both necessary and sufficient for endosomal recruitment. As in the case of full-length Doa4 (Luhtala and Odorizzi, 2004), localization of Doa4 -GFP to endosomes was dependent upon Bro1 (Figure 1D), and this region co-immunoprecipi- tated with Bro1 from yeast cell lysates (Figure 1E). Figure 1 The N terminus of Doa4 is necessary and sufficient for The catalytic domain of Doa4 is specifically required Bro1-mediated localization to endosomes. (A–D) Fluorescence and for cargo deubiquitination and sorting N CAT DIC microscopy of Doa4 -GFP, Doa4-GFP, and Doa4 -GFP. FM4- To determine whether endosomal localization is sufficient to 64 is a lipophilic stain that specifically labels E compartments and confer substrate specificity, we tested if the catalytic domain vacuolar membranes. Arrowheads indicate class E compartments that do (closed arrowheads) or do not (open arrowheads) colocalize of a different UBP was functional in the MVB pathway when with GFP. Scale bar, 2.5mm. (E) Native anti-HA immunoprecipita- fused to the Doa4 N terminus. Ubp5 is the UBP in yeast most tion followed by anti-HA or anti-Bro1 immunoblotting of total lysate closely related to Doa4 (Figure 2A), but GFP fused to full- versus bound proteins. The GFP fusions in (A–D) and the HA fusion length Ubp5 (Ubp5-GFP) failed to localize to endosomes in (E) were expressed from single copies integrated into the genome under the control of the endogenous DOA4 promoter. (Figure 2B), consistent with Ubp5 having no functional role in MVB sorting. As expected, appending the Doa4 N-terminal region to the Ubp5 catalytic domain led to endosomal loca- N CAT lization of the resulting fusion protein (Doa4 -Ubp5 -GFP; activity despite being recruited to the site of Doa4 function at endosomal membranes. Figure 2C). However, GFP-tagged carboxypeptidase S (GFP- CPS), a cargo protein normally sorted into the vacuole lumen via the MVB pathway (Odorizzi et al, 1998), was missorted to A YPxL motif in the catalytic domain of Doa4 mediates N CAT the vacuole membrane in cells expressing Doa4 -Ubp5 in binding to Bro1 place of wild-type Doa4, similar to the mislocalization of The specific requirement for the Doa4 catalytic domain in the C571S GFP-CPS in doa4 cells (Figure 2D), which express a MVB pathway led us to compare its amino-acid sequence catalytically inactive Doa4 mutant. In addition, the Doa4 - with that of the Ubp5 catalytic domain. Although the two CAT Ubp5 chimera was unable to deubiquitinate CPS in vivo sequences are B60% identical, Doa4 contains a YPFL se- (Figure 2E). Altogether, these observations indicate that the quence (amino acids 826–829) versus YPYS at the corre- Ubp5 catalytic domain cannot substitute for Doa4 enzymatic sponding position in Ubp5 (Figure 2A). The YPFL in Doa4 &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 10 2007 2455 | | Doa4-mediated deubiquitination at endosomes C Richter et al Figure 2 MVB cargo sorting and deubiquitination specifically require the Doa4 catalytic domain. (A) Schematic representations of Doa4, N CAT Ubp5, and the Doa4 -Ubp5 chimera. Amino-acid identity between Doa4 and Ubp5 is indicated. Fluorescence and DIC microscopy of Ubp5- N CAT GFP (B) or Doa4 -Ubp5 -GFP (C). Arrowheads indicate class E compartments that do (closed arrowheads) or do not (open arrowheads) colocalize with GFP. (D) Fluorescence and DIC microscopy of GFP-CPS. Scale bar, 2.5 mm (B–D). (E) Denatured anti-CPS immunoprecipitations followed by anti-Ub or anti-CPS immunoblotting. Doublet bands represent differentially glycosylated forms of CPS. The GFP fusions in (B, C) were expressed from single copies integrated into the genome under the control of the endogenous DOA4 promoter. matches the consensus sequence in viral proteins, YPxL, Alix binds directly to the YPxL consensus sequence in Gag which binds Alix, the mammalian ortholog of Bro1 (Martin- protein subunits of HIV-1, EIAV, and MuLV (Martin-Serrano Serrano et al, 2003; Strack et al, 2003; Segura-Morales et al, et al, 2003; Strack et al, 2003; Segura-Morales et al, 2005). 2005). Indeed, the YPFL sequence was required for Doa4 Therefore, we used a yeast two-hybrid assay to test whether function in the MVB pathway. When this motif was changed Bro1 binds the YPFL motif in Doa4. As a positive control, we AAFA to AAFA by site-directed mutagenesis (doa4 ), deubiqui- used Snf7, which binds directly to the ‘Bro1 domain’ of Bro1 tination of CPS was defective (Figure 3B), and GFP-CPS was (Figure 4A) (Kim et al, 2005). Accordingly, Snf7 interacted mislocalized to the vacuole membrane (Figure 3A). Pulse– with both full-length Bro1 and the Bro1 domain (amino acids chase metabolic labeling followed by anti-GFP immuno- 2–387) but not with the remaining C-terminal fragment of precipitation confirmed that the cleavage of GFP from CPS, Bro1 (amino acids 388–844; Figure 4B). The catalytic domain which occurs following delivery of MVB vesicles into the of Doa4 also bound full-length Bro1 but did not interact with vacuole lumen and accurately reflects the proteolytic matura- the Bro1 domain. Instead, the Doa4 catalytic domain bound 388–844 tion of native CPS (Odorizzi et al, 1998), was quantitatively Bro1 (Figure 4B). Because the Doa4 N terminus was C571S AAFA reduced to a similar extent in doa4 and doa4 cells removed to preclude the possibility of Bro1 binding it in the compared with cleavage of GFP-CPS in wild-type cells two-hybrid assay, Bro1 interacts with the Doa4 catalytic (Figure 3C). The extent of cleavage was not significantly domain independent of the endosomal localization region C571S AAFA 388–844 different in doa4 and doa4 cells expressing lower of Doa4. Binding of Bro1 to the Doa4 catalytic domain levels of GFP-CPS (Supplementary Figure S2), nor was the was abolished, however, when the YPFL motif in Doa4 was defect in cleavage rescued upon increased Ub expression changed to AAFL (Figure 4B). Moreover, the Doa4 YPFL motif (Supplementary Figure S3), which agrees with recent work conforms to the YPxL consensus sequence because individual showing that the depleted pools of free Ub, characteristic of mutation of Y ,P ,orL , but not F , disrupted the 826 827 829 828 388–844 doa4 mutant cells, do not account for MVB sorting defects in interaction with Bro1 (Figure 4C). In addition, V and cells lacking Doa4 function (Nikko and Andre, 2007). L in Doa4 are required for this interaction, whereas I is 830 825 C571S AAFA However, GFP-CPS cleavage in doa4 and doa4 cells dispensable. was not completely blocked, as was observed in pep4D prb1D cells lacking vacuolar protease activity (Figure 3C). Thus, the Truncation of Bro1 disables it from binding the Doa4 efficiency of GFP-CPS sorting is reduced in the absence of catalytic domain and results in Doa4-specific MVB Doa4 function but not completely defective, as seen pre- pathway defects viously in cells lacking the components of the core MVB To map the YPxL-interacting region, we screened a collection sorting machinery of class E Vps proteins, including Vps4 of mutant bro1 alleles for their ability to phenocopy the MVB (vps4D; Figure 3C) (Reggiori and Pelham, 2001; Babst et al, pathway defect caused by loss of Doa4 function. A hallmark 2002). The YPFL motif was not required for recruitment of phenotype caused by deletion of the DOA4 gene is the Doa4 to endosomes (Figure 3D), indicating that, like the selective block in sorting of Ub-dependent MVB cargoes N CAT AAFA Doa4 -Ubp5 chimera (Figure 2), Doa4 is unable to such as CPS, whereas sorting of Ub-independent cargoes function in the MVB pathway despite being recruited to the such as Sna3 is unaffected (Reggiori and Pelham, 2001). In site of Doa4 activity on endosomal membranes. contrast, deletion of any class E VPS gene, including BRO1, 2456 The EMBO Journal VOL 26 NO 10 2007 &2007 European Molecular Biology Organization | | Doa4-mediated deubiquitination at endosomes C Richter et al Figure 3 The Doa4 YPxL motif is necessary for cargo sorting and deubiquitination. (A) Fluorescence and DIC microscopy of GFP-CPS. (B) Denatured anti-CPS immunoprecipitations followed by anti-Ub and anti-CPS immunoblotting. Doublet bands represent differentially glycosylated forms of CPS. The slower migrating bands in the anti-Ub panel might represent more extensive Ub modification of CPS. (C) Quantification of GFP-CPS cleavage measured after anti-GFP immunoprecipitation from S-labeled cell extracts; P-valueso0.05 (*), 0.01 (**), AAFA or 0.001 (***). (D) Fluorescence and DIC microscopy of vps4D cells expressing the Doa4 -GFP fusion from a low-copy number plasmid under the control of the DOA4 promoter. Closed arrowheads indicate class E compartments that colocalize with GFP. Scale bar, 2.5 mm (A, D). blocks sorting of both Ub-dependent and -independent car- ability of Bro1 to recruit Doa4 to endosomes (Figure 5B). goes via the MVB pathway (Figure 5A) (Reggiori and Pelham, Together, these observations suggested that truncation of 2001). Although the general mechanism by which Bro1 the Bro1 C terminus prevents it from binding the YPxL function is required for sorting MVB cargoes is unknown, motif of Doa4. we reasoned that its interaction with Doa4 constitutes a The Doa4-specific MVB sorting defect caused by the bro1-2 subdivision of function specifically required for sorting mutation was further supported by three-dimensional elec- Ub-dependent MVB cargoes. Thus, a mutation in Bro1 that tron tomographic modeling. In wild-type yeast cells, MVBs prevents it from binding the YPxL motif of Doa4 but are spherical structures that have numerous lumenal vesicles otherwise has no adverse effect on Bro1 function should (Figure 5C and Supplementary Video S5). In contrast, dele- block the sorting of CPS but not Sna3. Indeed, the bro1-2 tion of the BRO1 gene dramatically alters the morphology of allele, which contains a premature stop codon in place of endosomes such that they consist of flattened cisternae-like codon 820 (Figure 4A), caused mislocalization of GFP-CPS structures within which lumenal vesicles are entirely absent to the vacuole membrane, but did not block sorting of (Figure 5D and Supplementary Video S6). This abnormal Sna3-GFP into the vacuole lumen (Figure 5A). Furthermore, endosomal morphology is a unique characteristic caused by bro1-2 impaired GFP-CPS cleavage to a similar extent as deletion of any class E VPS gene. Therefore, these aberrant AAFA observed in doa4 cells (Figure 3C and Supplementary structures are referred to as ‘class E compartments’ Figure S2). Yeast two-hybrid analysis confirmed that (Raymond et al, 1992; Rieder et al, 1996; Babst et al, 1997; the mutant Bro1-2 protein was unable to bind the Doa4 Odorizzi et al, 1998). Unlike bro1D cells, bro1-2 cells do not catalytic domain (Figure 4B), and fluorescence microscopy have class E compartments, but instead have spherical multi- indicated that the bro1-2 mutation had no effect on the vesicular endosomes (Figure 5E and Supplementary Video &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 10 2007 2457 | | Doa4-mediated deubiquitination at endosomes C Richter et al Figure 4 Bro1 interacts with the Doa4 YPxL motif. (A) Schematic diagram of Bro1 indicating the ‘Bro1 domain’, coiled-coil (CC), and proline- rich domain (PRD). The bro1-2 mutation replaces the glutamine at position 820 with a termination codon. (B, C) Yeast two-hybrid analysis of the interaction between Doa4 and Bro1. Bro1 fragments fused to the Gal4 activation domain are indicated with GAD-Bro1, including the CAT appropriate amino-acid residues. The LexA DNA-binding domain was fused to Snf7 from A. nidulans, Doa4 , and the indicated amino-acid CAT substitutions within Doa4 . C571S S7), similar to those observed in doa4 cells (Figure 5F sequence at the C-terminal tip of Bro1 is highly conserved and Supplementary Video S8). among its candidate orthologs in other fungal species The presence of lumenal MVB vesicles in bro1-2 and (Figure 6A). Therefore, we tested whether mutation of C571S 793–844 doa4 cells is consistent with both strains having a func- its C-terminal residues affected binding of Bro1 to CAT tional MVB pathway capable of sorting Sna3-GFP but not Doa4 . As shown in Figure 6B, this interaction was Ub-dependent cargoes such as GFP-CPS. However, closer disrupted upon substitution of four alanine residues in examination revealed another phenotype caused by the place of the PSVF sequence spanning amino acids 831–834 C571S 793–844(PSVF–AAAA) bro1-2 and doa4 mutations. The lumenal MVB vesicles of Bro1 (Bro1 ), or upon individual substitu- of both mutant strains were predominantly smaller than tion of alanine for P ,S ,V ,orF . Binding also did 831 832 833 834 MVB vesicles in wild-type cells (Figure 5G) even though the not occur upon mutation of R ,M ,Y ,orY , whereas 830 838 839 842 size of MVBs was unchanged (Supplementary Figure S4B). the interaction was unaffected by substitution of alanine in Although the molecular basis for larger lumenal MVB vesicles place of D ,E ,N ,S ,K ,S ,orS (Figure 6A 835 836 837 840 841 843 844 C571S being absent in bro1-2 and doa4 cells is not clear, this and data not shown). Consistent with this region of Bro1 observation provided further evidence that truncation of Bro1 binding to the YPxL motif in Doa4, cells expressing the PSVF–AAAA mimics the MVB pathway defects caused by loss of Doa4 bro1 allele were defective in deubiquitination and function. sorting of CPS (Figure 6C and D) and contained a predomi- Overexpression of Ub restored the formation of larger nance of small lumenal MVB vesicles (Supplementary Figure lumenal vesicles in cells lacking Doa4 activity (Supplemen- S4A and Supplementary Video S9). Thus, binding of the YPxL tary Figure S3C), but also caused distortions of the limiting motif in Doa4 by the conserved C terminus of Bro1 is required endosomal membrane (Supplementary Figure S3D), resulting for Doa4-mediated deubiquitination of MVB cargoes. in structures we recently characterized as vesicular tubular endosomes (VTEs) (Nickerson et al, 2006). VTEs exist in cells lacking expression of Did2, an adaptor protein that couples Bro1 stimulates deubiquitination by Doa4 Vps4 activity to ESCRT-III dissociation from endosomes. For To test directly the effect of Bro1 on deubiquitination, we CAT unknown reasons, the lumenal vesicles of VTEs in did2D isolated recombinant Doa4 from bacteria and analyzed its cells are unusually large (Nickerson et al, 2006). Thus, it is enzymatic activity in vitro in the presence or absence of 388–844 CAT unclear whether the increased lumenal vesicle size caused by recombinant Bro1 . Incubation of GST-Doa4 with overexpression of Ub in cells lacking Doa4 function is simply Ub fused to 7-amino-4-methylcoumarin (Ub-AMC) resulted due to replenished pools of free cellular Ub or, instead, a in a low rate of hydrolysis, as measured by emission of AMC 388–844 manifestation of the VTE morphology. fluorescence (Figure 7A). Addition of His -Bro1 to the reaction dramatically enhanced the rate of hydrolysis, but A conserved C-terminal motif in Bro1 is required for its this stimulatory effect was lost when the PSVF sequence in interaction with the catalytic domain of Doa4 Bro1 was mutated (Figure 7A), confirming that this motif is Based on the inability of the Bro1-2 protein to bind the Doa4 essential for binding to the Doa4 catalytic domain and catalytic domain, we hypothesized that a C-terminal fragment stimulation of deubiquitination. Maximal stimulation of 388–844 of Bro1 would be sufficient for this interaction to occur. Ub-AMC hydrolysis was reached when His -Bro1 was CAT Indeed, the last 52 amino acids of Bro1 (residues 793–844) present at 1- to 2-fold amounts relative to GST-Doa4 CAT interacted with Doa4 (Figure 4B). The amino-acid (Figure 7B), indicating a stoichiometric relationship. 2458 The EMBO Journal VOL 26 NO 10 2007 &2007 European Molecular Biology Organization | | Doa4-mediated deubiquitination at endosomes C Richter et al Figure 5 The C terminus of Bro1 is specifically required for Ub-dependent cargo sorting. (A) Fluorescence and DIC microscopy of GFP-CPS and Sna3-GFP. (B) Fluorescence and DIC microscopy of Doa4-GFP. Arrowheads indicate E compartments that do (closed arrowheads) or do not C571S (open arrowheads) colocalize with GFP. Scale bar, 2.5mm (A, B). (C–F) EM showing MVBs in wild-type, bro1-2, and doa4 cells, and the class E compartment in bro1D cells. Vesicles in tomographic models have diameters indicated by green (18–23 nm), purple (24–29 nm), pink (30–35 nm), red (36–47 nm). V¼ vacuole. Scale bar, 0.1mm(G) Distribution of MVB vesicle diameters from wild-type (n¼ 330), bro1-2 C571S C571S (n¼ 265), and doa4 cells (n¼ 371). Mean vesicle diameters of bro1-2 and doa4 cells differ significantly from that of wild-type cells (Po0.0001). 388–844 To determine whether Bro1 increases the catalytic Discussion activity and/or substrate affinity of Doa4, we generated Michaelis–Menten curves by testing the reaction velocity at Bimodal regulation of localization and activation is emerging varying Ub-AMC concentrations. As shown in Figure 7C, as a paradigm for controlling the specificity of enzymes that CAT 388–844 GST-Doa4 incubated with His -Bro1 yielded a V function in multiple cellular processes (Bhattacharyya et al, 6 max of Ub-AMC hydrolysis at 75.16 nmole/min, resulting in a K 2006; Chen and Kass, 2006; Dard and Peter, 2006). Doa4 is a CAT of 0.56 mM. However, the reaction velocity for GST-Doa4 UBP in S. cerevisiae that removes Ub from membrane pro- alone was linearly related to substrate concentration teins targeted into the MVB pathway (Dupre and (Figure 7C), which precluded determination of the V and Haguenauer-Tsapis, 2001; Katzmann et al, 2001; Losko max K . Nevertheless, we infer from Figure 7C that the K of et al, 2001), and from soluble proteins targeted to protea- M M CAT Ub-AMC hydrolysis by GST-Doa4 alone is significantly somes (Papa et al, 1999). In addition, Doa4 activity has been CAT higher than the K of GST-Doa4 in the presence of His - implicated in the coordination of DNA replication (Singer M 6 388–844 Bro1 , suggesting that Bro1 increases the affinity of et al, 1996) and the DNA damage response (Fiorani et al, Doa4 for its substrate. 2004). Our results indicate that Bro1 both recruits Doa4 to &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 10 2007 2459 | | Doa4-mediated deubiquitination at endosomes C Richter et al Figure 6 The Bro1 PSVF motif interacts with the Doa4 YPxL motif. (A) Multiple sequence alignment of the C termini of Bro1 homologs from several fungal species. Arrowheads indicate Bro1 residues CAT required for interaction with Doa4 by two hybrid. (B) Yeast two-hybrid analysis of the interaction between Doa4 and Bro1. (C) Fluorescence and DIC microscopy of GFP-CPS. Scale bar, 2.5mm. (D) Anti-CPS immunoblot of whole-cell lysates. TheB9 kDa shift in CPS corresponds to its monoubiquitinated form. late endosomes and stimulates its catalytic activity, thereby exerting dual modes of regulation to control the specificity of Doa4 function in the MVB pathway. Site-specific activation by Bro1 ensures that Doa4 is active during its transient associa- Figure 7 Bro1 stimulates Doa4-mediated deubiquitination in vitro. tion with endosomes and implies that the location and timing (A) Measurement of fluorescence generated by deubiquitination of Ub-AMC (0.4mM; l : 380 nM, l : 440 nM) using 50 nM of Doa4 activity outside of the MVB pathway is also regulated ex em 406–926 388–844 GST-Doa4 with or without 100 nM His -Bro1 or His - 6 6 to prevent nonspecific protein deubiquitination. Dual-regula- 388–844(PSVF–AAAA) Bro1 . All Bro1 and Doa4 proteins were purified tory mechanisms may similarly function in other cases of from E. coli and reactions were run for 7 min in triplicate. UBP-mediated deubiquitination. The catalytic activity of Measurements were also taken in the absence of either enzyme or substrate to control for aberrant deubiquitination or fluorescence, Ubp6 is stimulated by its recruitment to proteasomes respectively. (B) Doa4-mediated Ub-AMC hydrolysis under varying (Leggett et al, 2002), as is the activity of Ubp8 upon its 388–844 388–844(PSVF–AAAA) concentrations of His -Bro1 or His -Bro1 . 6 6 assembly into the SAGA histone acetyltransferase complex 406–926 GST-Doa4 and Ub-AMC concentrations were kept constant (Lee et al, 2005). In addition, the catalytic activity of AMSH, at 50 nM and 0.4 mM, respectively. The deubiquitination rate 406–926 was normalized to that of GST-Doa4 in the absence of which belongs to the JAMM metalloprotease family of DUBs, 388–844 406–926 His -Bro1 .(C) Ub-AMC hydrolysis by GST-Doa4 is stimulated upon binding to the endosomal sorting factor (50 nM) under varying substrate concentrations, with or without STAM (McCullough et al, 2006). 388–844 His -Bro1 (100 nM). All data points are represented by The finding that Bro1 reduces the K of deubiquitination is M mean7s.e.m. consistent with a model in which it enhances substrate binding by Doa4, although the mechanism by which this catalytic autoinhibitory C terminus (Yao et al, 2006). occurs is unknown. In mammalian cells, Adrm1 reduces the Although Uch37 belongs to the Ub C-terminal hydrolase K of Uch37-mediated deubiquitination by binding its non- (UCH) family of DUBs, structural analyses indicate that the 2460 The EMBO Journal VOL 26 NO 10 2007 &2007 European Molecular Biology Organization | | Doa4-mediated deubiquitination at endosomes C Richter et al catalytic cores of UCH and UBP enzymes are very similar in this manner by binding the YPxL motif of Doa4 was guided (Johnston et al, 1999; Hu et al, 2002). The ability of Adrm1 to by previous work showing that the mammalian ortholog of relieve autoinhibition by binding the non-catalytic C-terminal Bro1, Alix, binds a YPxL consensus sequence in Gag protein region of Uch37, however, is distinct from the mechanism by subunits encoded by HIV-1, EIAV, and MuLV. As a conse- which Bro1 binds directly to the catalytic domain of Doa4 to quence, Alix is recruited to the site of viral assembly to stimulate its activity. Recent structural analysis of UBPY, the facilitate budding of infectious virions from host cells mammalian UBP most closely related to Doa4, has revealed it (Puffer et al, 1997; Martin-Serrano et al, 2003; Strack et al, to be autoinhibited by b-strands and loop segments in its 2003; Segura-Morales et al, 2005). Like MVB cargoes, viral catalytic domain that block access to the Ub-binding site, and Gag proteins are ubiquitinated, but it is unclear whether Ub only after these elements are displaced, is UBPY capable of modification is relevant to viral budding (Morita and substrate binding and deubiquitination (Avvakumov et al, Sundquist, 2004). However, it is noteworthy that transplanta- 2006). Movement of these features in UBPY was proposed to tion of the YPxL motif into recombinant viral constructs be under the control of helix 12, which might act as a hinge. reduces the amount of Ub-conjugated Gag (Martin-Serrano Alignment of the UBPY and Doa4 catalytic domain sequences et al, 2004), suggesting that Alix subsequently results in positions the YPxL motif of Doa4 adjacent to helix 12 of UBPY recruitment of a DUB in a way similar to recruitment of (Avvakumov et al, 2006), raising the possibility that binding Doa4 by Bro1. of Bro1 to this site induces a structural rearrangement that YPxL motifs also mediate protein interactions with homo- provides the catalytic site of Doa4 access to ubiquitinated logs of Bro1 that have no functional connection to the MVB MVB cargoes. However, this interaction is likely to be tran- pathway. Rim101 in S. cerevisiae and PacC in Aspergillus sient, as we cannot detect stable binding between GST- nidulans are YPxL-containing transcription factors that un- CAT 388–844 Doa4 and His -Bro1 in vitro (C Richter and G dergo proteolytic cleavage and activation upon interacting Odorizzi, unpublished observations). with the Bro1 homologs Rim20 and PalA, respectively The role of the N terminus of Doa4 in its endosomal (Xu and Mitchell, 2001; Vincent et al, 2003). Although the recruitment is consistent with a modular principle of UBPs C-terminal PSVF-containing sequence in Bro1 is conserved in which their non-catalytic domains mediate localization to among the predicted functional orthologs of Bro1 in fungal specific subcellular sites of function (Leggett et al, 2002; Hu species, it is not found in Rim20 and PalA. Similarly, the C et al, 2005). Recent work identified four conserved amino- termini of Bro1 and Alix share little sequence homology, and acid motifs in the N-terminal region of Doa4 that are required the region of Alix that binds viral YPxL sequences is located for its localization to endosomes (Amerik et al, 2006). in the middle of the protein between its Bro1 domain and However, based on analysis of an overexpressed Doa4-GFP proline-rich region (Fisher et al, 2007; Lee et al, 2007). fusion protein, the same study concluded that Bro1 is dis- Therefore, any similarity between the PSVF motif of Bro1 pensable for endosomal localization of Doa4 (Amerik et al, and the YPxL-binding regions of Alix, Rim20, and PalA might 2006). Although Bro1 physically associates with the N-term- only be apparent at the level of tertiary structure. inal endosomal localization region of Doa4 in cell lysates Mutations that disrupt the PSVF motif in Bro1 phenocopy (this study) and is required for the endosomal localization of the loss of Doa4 function by blocking the sorting of CPS, a Doa4-GFP expressed at physiologically normal levels (this Ub-dependent MVB cargo, whereas the sorting of Sna3, a Ub- study and Luhtala and Odorizzi, 2004), we have also found independent cargo, continues normally. Accordingly, bro1-2 PSVF–AAAA C571S that, when overexpressed, Doa4-GFP localizes to endosomes and bro1 mutant cells both resemble doa4 cells in the absence of Bro1 (C Richter and G Odorizzi, unpub- in that they have MVBs rather than the class E compartments lished observations). It is likely, therefore, that another that are characteristically observed upon deletion of BRO1 or component on endosomal membranes binds Doa4, but this any other class E VPS gene (Raymond et al, 1992; Rieder et al, factor, alone, is insufficient to effectively concentrate Doa4 at 1996; Babst et al, 1997; Odorizzi et al, 1998). Defining how endosomes under the normal conditions of low Doa4 expres- class E Vps proteins coordinate both MVB cargo sorting and sion. Such a factor may also stabilize the interaction between lumenal vesicle formation is a major challenge in under- Bro1 and the Doa4 N terminus because their binding is standing the molecular mechanism of MVB function. Our undetectable in the yeast two-hybrid assay (C Richter and G results clearly indicate that a division of labor exists for Bro1 Odorizzi, unpublished observations). in these processes. Insight into the specific role that Bro1 has In addition to causing MVB sorting defects, loss of Doa4 in forming lumenal MVB vesicles might be gleaned from function renders cells hypersensitive to canavanine, a cyto- studies indicating that its mammalian ortholog, Alix, binds toxic arginine analog (Papa and Hochstrasser, 1993). The lysobisphospatidic acid (LBPA) to regulate the dynamic abil- canavanine hypersensitivity can be rescued by fusion of the N ity of this lipid to promote either fission or fusion of lumenal terminus of Doa4 to the catalytic domain of its most closely MVB membranes (Matsuo et al, 2004; Le Blanc et al, 2005). related yeast homolog, Ubp5 (Amerik et al, 2006). However, However, a similar role for Bro1 in regulating membrane we found that MVB cargo deubiquitination and sorting could dynamics likely differs mechanistically because LBPA has not not be rescued by an identical Doa4-Ubp5 fusion protein (nor been detected in yeast. could GFP-CPS cleavage; C Richter and G Odorizzi, unpub- The range of MVB vesicle diameters observed in wild-type lished observation), indicating that canavanine sensitivity is yeast cells sharply contrasts with the predominantly small caused by a defect in Doa4 function unrelated to the MVB vesicles observed in cells lacking Doa4 activity. How lumenal pathway. The inability of the Ubp5 catalytic domain to MVB vesicle size correlates with Doa4 function is unknown. substitute for that of Doa4 led us to speculate that an If deubiquitination has a direct role in MVB vesicle size endosome-associated factor might function specifically to determination, it might be coupled to activation of a mole- stimulate Doa4 activity. Our discovery that Bro1 functions cular machinery that controls the formation of larger vesicles &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 10 2007 2461 | | Doa4-mediated deubiquitination at endosomes C Richter et al (Odorizzi et al, 2003) and mouse anti-HA monoclonal antibodies within which Ub-dependent cargoes such as CPS are selec- (Covance). For pulse–chase metabolic labeling, 5 A equivalents tively packaged, while a separate machinery might operate of yeast cells were incubated with 100 mCi S-labeled methionine/ independently to form smaller vesicles that transport cysteine for 10 min at 301C, followed by the addition of chase Ub-independent cargoes such as Sna3. Alternatively, lumenal mixture (5 mM methionine, 1 mM cysteine, and 0.2% yeast extract) for 0 or 30 min. Afterward, cells were precipitated by the addition of MVB vesicle size might be determined by the influx of 10% (vol/vol) trichloroacetic acid, cell lysates were prepared under cargoes into the pathway. Most yeast cargoes are dependent denaturing conditions, and immunoprecipitations of GFP-CPS were upon the cycle of ubiquitination and deubiquitination and, as performed as previously described (Katzmann et al, 2001; Luhtala such, are blocked from entering the MVB pathway in cells and Odorizzi, 2004) using anti-GFP polyclonal antiserum (Odorizzi et al, 1998). Immunoprecipitates were resolved by SDS–PAGE and lacking Doa4 activity (Loayza and Michaelis, 1998; Dupre exposed to a storage phosphor screen (GE Healthcare), and then and Haguenauer-Tsapis, 2001; Reggiori and Pelham, 2001; developed by a Storm Phosphorimager (GE Healthcare) and Urbanowski and Piper, 2001). Deubiquitination might, there- TM quantified using ImageQuant TL (GE Healthcare). All samples fore, have no direct influence on MVB vesicle formation, with were tested in triplicate and the Prism software (Graphpad Software) was used for statistical analysis. smaller vesicles providing adequate surface area to accom- modate a decreased cargo load in the absence of Doa4 Isolation of the bro1-2 allele function. BRO1 was PCR-amplified using Taq polymerase (Invitrogen) under error-prone conditions (Guthrie and Fink, 2002) to generate a library of random mutant bro1 alleles that were pooled and Materials and methods cotransformed into yeast strain KGY1 (Luhtala and Odorizzi, 2004) together with pGO221 (pRS416 containing the BRO1 gene) Yeast strains and plasmid construction that had been linearized by EcoRI/HindIII digestion. In vivo For information, see Supplementary data. homologous recombination yielded 410 000 colonies of KGY1, each of which contained one repaired plasmid encoding a single Fluorescence microscopy bro1 allele. KGY1 expresses the carboxypeptidase Y-invertase Strains were grown to logarithmic phase at 301C in synthetic reporter fusion protein, which is secreted upon loss of Bro1 medium before observation at room temperature using a Zeiss function (Luhtala and Odorizzi, 2004). Thus, colonies of KGY1 Axioplan 2 microscope with an NA 1.40 oil-immersion objective transformed with plasmids encoding non-functional mutant bro1 (Carl Zeiss MicroImaging Inc.). Fluorescence and differential alleles were identified based on the detection of secreted invertase interference contrast (DIC) images were acquired with a Cooke activity using a colorimetric agar overlay assay (Darsow et al, 2000; Sensicam digital camera (Applied Scientific Instruments Inc.) and Luhtala and Odorizzi, 2004). Whole-cell lysates prepared from processed using Slidebook (Intelligent Imaging Innovations) and clones secreting invertase were analyzed by SDS–PAGE and Western Photoshop 7.0 software (Adobe). Cells were stained with FM4-64 blotting using rabbit anti-Bro1 antiserum to eliminate plasmids not (Molecular Probes Inc.) using a pulse–chase procedure as described expressing Bro1. The remaining plasmids were isolated from yeast previously (Odorizzi et al, 2003). and subjected to DNA sequence analysis. Among the mutant bro1 alleles isolated by this procedure, the bro1-2 allele was the only one High-pressure freeze substitution and electron tomography that was capable of sorting Sna3-GFP but not GFP-CPS via the MVB Cells were high-pressure frozen, freeze-substituted with 0.1% pathway. uranyl acetate, 0.25% glutaraldehyde, anhydrous acetone at 901C, embedded in Lowicryl HM20, and polymerized under UV Yeast two-hybrid at 501C (Winey et al, 1995). Semi-thick sections (200 nm) were CTY10.5d was transformed with GAD and LEXA fusions (Vincent placed on rhodium-plated formvar-coated copper slot grids and et al, 2003), patched onto agar medium, allowed to grow for 3 days, mapped on a Phillips CM10 TEM at 80 kV. Dual tilt series images then lysed by exposure to chloroform vapor for 30 min before were collected from þ 601 to 601 with 11 increments at 200 kV overlay with 0.6% agar containing Z-buffer (60 mM Na HPO , 2 4 using a Tecnai 20 FEG (FEI). Tomograms were imaged at  29 000 . . 40 mM NaH PO H 0, 10 mM KCl, 1 mM MgSO 7H 0, 0.2% 2 4 2 4 2 with a 0.77 nm pixel (binning 2). Sections were coated on both sides b-mercaptoethanol, and 0.67 mg/ml X-gal). Blue/white analysis with 15-nm fiducial gold for reconstruction of back projections was performed after 6 h to identify interactions. using IMOD software (Kremer et al, 1996). 3dmod software was used for mapping structure surface areas. Mean z-scale values for In vitro deubiquitination assays sections were within 3%. Best-fit sphere models were used to 406926 388–844 388–844(PSVF–AAAA) GST-Doa4 , His -Bro1 , and His -Bro1 6 6 measure vesicle diameters to the outer leaflet of membrane bilayers. were expressed in Escherichia coli BL21-CodonPlus (DE3) cells IMOD calculated limiting membrane surface areas using three- (Stratagene) by induction with 0.5 mM isopropyl-b-D-thiogalacto- dimensional mesh structures derived from closed contours drawn side at 201C for 18 h and purified using glutathione–Sepharose (GE each 3.85 nm using imodmesh. For quantitation, vesicles from 12– Healthcare) or TALON metal affinity resin (Clontech). For deubi- 13 random MVBs were measured from 4–5 cells of each strain and 406–926 quitination reactions, 50 nM GST-Doa4 was incubated with or analyzed using the Gaussian curve fit of the Prism software 388–844 388–844(PSVF–AAAA) without His -Bro1 , or His -Bro1 , in reaction 6 6 (GraphPad Software). buffer (50 mM HEPES pH 7.5, 2 mM DTT, and 0.1 mg/ml BSA) for 30 min at 251C in 96-well plates. Reactions were initiated by the Immunoprecipitation and Western blotting addition of Ub-AMC (Boston Biochem) and analyzed by a Tecan Denatured immunoprecipitations to detect Ub-CPS were performed Safire II fluorescence plate reader (l : 380 nM, l : 440 nM), which ex em as described previously (Katzmann et al, 2001; Luhtala and maintained the reaction at 251C. All samples were tested in Odorizzi, 2004). The 0.5 A equivalents were resolved by SDS– 600 triplicate and Prism (GraphPad Software) was used for statistical PAGE, transferred to nitrocellulose, and analyzed by Western blot analysis and nonlinear fit to Michaelis–Menten kinetics. using rabbit anti-CPS polyclonal antiserum (Cowles et al, 1997), anti-Ub monoclonal antibodies (Zymed), and anti-phosphoglyce- Supplementary data rate kinase (PGK) monoclonal antibodies (Invitrogen). For native Supplementary data are available at The EMBO Journal Online 1–560 immunoprecipitations of Doa4 -TEV-HA, yeast lysates prepared (http://www.embojournal.org). as previously described (Luhtala and Odorizzi, 2004) were incubated with mouse anti-HA monoclonal antibodies (Covance) and protein G–Sepharose beads (GE Healthcare) for 2 h at 41C, after Acknowledgements which the beads were collected by centrifugation and washed (Luhtala and Odorizzi, 2004), and 10 A equivalents of We thank Sarah Altschuler and Megan Wemmer (University of immunoprecipitates or 0.5 A equivalents of total lysate were Colorado) for plasmid and yeast strain construction, Doug Burch resolved by SDS–PAGE, transferred to nitrocellulose, and analyzed (University of Colorado) for yeast strain construction and electron by Western blot using rabbit anti-Bro1 polyclonal antiserum microscopy, Amy Palmer (University of Colorado) for the use of the 2462 The EMBO Journal VOL 26 NO 10 2007 &2007 European Molecular Biology Organization | | Doa4-mediated deubiquitination at endosomes C Richter et al Tecan Safire II fluorescence plate reader, Olivier Vincent (Centro de and Eric Cooper (Johns Hopkins University) for advice on DUB Investigaciones Biologicas del CSIC) for providing the yeast two- kinetic analysis. 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Dual mechanisms specify Doa4‐mediated deubiquitination at multivesicular bodies

Richter, Caleb; West, Matthew; Odorizzi, Greg
The EMBO Journal , Volume 26 (10) – May 16, 2007
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Abstract

The EMBO Journal (2007) 26, 2454–2464 & 2007 European Molecular Biology Organization All Rights Reserved 0261-4189/07 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies where it deubiquitinates integral membrane proteins ubiqui- Caleb Richter, Matthew West tinated on their cytosolic domains. Monoubiquitination (or, and Greg Odorizzi* in some cases, polyubiquitination with a short chain of 2–3 Department of Molecular, Cellular, and Developmental Biology, Ub subunits) targets membrane proteins into lumenal MVB University of Colorado, Boulder, CO, USA vesicles formed by invagination of the late-endosomal mem- brane (Dupre and Haguenauer-Tsapis, 2001; Katzmann et al, Doa4 is a ubiquitin-specific protease in Saccharomyces 2001; Reggiori and Pelham, 2001; Urbanowski and Piper, cerevisiae that deubiquitinates integral membrane pro- 2001). Lumenal MVB vesicles and their cargoes are ultimately teins sorted into the lumenal vesicles of late-endosomal delivered into the hydrolytic interior of the vacuole upon multivesicular bodies (MVBs). We show that the non- fusion of the limiting endosomal membrane with the vacuo- catalytic N terminus of Doa4 mediates its recruitment to lar membrane. Doa4 ensures that Ub is recovered from endosomes through its association with Bro1, which is one cargoes before their enclosure within lumenal vesicles of several highly conserved class E Vps proteins that (Dupre and Haguenauer-Tsapis, 2001; Katzmann et al, 2001; comprise the core MVB sorting machinery. In turn, Bro1 Losko et al, 2001). Recruitment of Doa4 to late endosomes directly stimulates deubiquitination by interacting with a involves Bro1, one of several highly conserved ‘class E’ Vps YPxL motif in the catalytic domain of Doa4. Mutations in proteins that comprise the core machinery required for MVB either Doa4 or Bro1 that disrupt catalytic activation of cargo sorting and lumenal vesicle formation (Luhtala and Doa4 impair deubiquitination and sorting of MVB cargo Odorizzi, 2004). Bro1-mediated recruitment of Doa4 occurs proteins and lead to the formation of lumenal MVB vesi- downstream of cargo recognition, which is executed by Ub- cles that are predominantly small compared with the binding class E Vps proteins of the endosomal sorting com- vesicles seen in wild-type cells. Thus, by recruiting Doa4 plexes required for transport (ESCRTs) (Hurley and Emr, to late endosomes and stimulating its catalytic activity, 2006). Bro1 fulfills a novel dual role in coordinating deubiquiti- In mammalian cells, ubiquitinated membrane proteins are nation in the MVB pathway. similarly recognized by orthologs of the class E Vps machin- The EMBO Journal (2007) 26, 2454–2464. doi:10.1038/ ery and deubiquitinated by UBPY, the mammalian ortholog of sj.emboj.7601692; Published online 19 April 2007 Doa4 (Mizuno et al, 2005; Row et al, 2006). Mammalian class Subject Categories: membranes & transport E Vps proteins are also commandeered by many enveloped Keywords: deubiquitination; endosomes; multivesicular viruses to facilitate budding of infectious virions from host bodies; protein sorting cells, which is a Ub-dependent process topologically equiva- lent to the budding of lumenal MVB vesicles (Morita and Sundquist, 2004). Class E Vps proteins are recruited to the site of viral budding through interaction with ‘late domain’ Introduction motifs in virally encoded proteins. Alix, the mammalian ortholog of Bro1, binds the YPxL late domain motif in the Deubiquitinating enzymes (DUBs) catalyze removal of ubi- Gag subunit of human immunodeficiency virus-1 (HIV-1), quitin (Ub) from proteins targeted for degradation, thereby equine infectious anemia virus (EIAV), and murine leukemia replenishing the pool of free cellular Ub. DUBs also function virus (MuLV) (Martin-Serrano et al, 2003; Strack et al, 2003; in non-proteolytic processes such as cleavage of inactive Segura-Morales et al, 2005). Transplantation of YPxL into a poly-Ub translational fusions and deubiquitination of his- recombinant retrovirus results in enhanced deubiquitination tones, which regulate chromosome condensation and tran- of Gag (Martin-Serrano et al, 2004), suggesting that Alix scriptional silencing during mitosis (Kim et al, 2003; Amerik mediates recruitment of UBPY (or a different DUB) in a and Hochstrasser, 2004). The Ub-specific protease (UBP) manner analogous to recruitment of Doa4 by Bro1. family encompasses the majority of DUBs identified by In addition to its role in the MVB pathway, Doa4 functions sequence homology (Clague and Urbe, 2006). Many UBPs in removal of poly-Ub from soluble proteins targeted for are coupled to specific cellular processes, but the factors that degradation at proteasomes (Papa et al, 1999) and is impli- determine their functional specificities are largely uncharac- cated in the control of DNA replication and repair (Singer terized. et al, 1996; Fiorani et al, 2004). Regulating Doa4 specificity in Doa4 is a UBP in Saccharomyces cerevisiae that functions in multiple cellular processes is likely to be dependent upon the multivesicular body (MVB) protein sorting pathway, mechanisms that control its subcellular localization. However, we report that, alone, localization of Doa4 to *Corresponding author. Department of Molecular, Cellular, and Developmental Biology, University of Colorado, 347 UCB, Colorado endosomes is insufficient for specifying deubiquitination of Avenue, Boulder, CO 80309, USA. Tel.: þ 1 303 735 0179; MVB cargo proteins. Doa4 must also be activated upon its Fax: þ 1 303 492 7744; E-mail: [email protected] recruitment to endosomal membranes. Bro1 fulfills both roles by associating with the non-catalytic N-terminal region Received: 29 November 2006; accepted: 29 March 2007; published online: 19 April 2007 of Doa4 to mediate its localization to endosomes and by 2454 The EMBO Journal VOL 26 NO 10 2007 &2007 European Molecular Biology Organization | | Doa4-mediated deubiquitination at endosomes C Richter et al interacting with the catalytic domain of Doa4 to stimulate deubiquitination. Enzymatic activation of Doa4 occurs through binding of a conserved proline-based sequence located near the C terminus of Bro1 to a YPxL motif in Doa4. Mutations that prevent Bro1 from activating Doa4 disrupt deubiquitination and sorting of MVB cargo proteins. Thus, Bro1 coordinates Doa4 function and specificity by recruiting Doa4 to the MVB and acting as a cofactor to enhance deubiquitination. Results Bro1 associates with the N-terminal region of Doa4 to mediate its recruitment to endosomes Doa4 is one of 16 UBPs in S. cerevisiae, all of which contain highly conserved C-terminal catalytic domains coupled to N-terminal regions with low similarity among one another (Amerik and Hochstrasser, 2004). The N-terminal regions of UBPs are thought to determine substrate specificity, poten- tially by mediating localization of each UBP to a distinct subcellular site of function (Kim et al, 2003). Indeed, GFP fused to amino acids 1–560 of Doa4 (Doa4 -GFP; Figure 1A) localized to FM4-64-stained endosomes as efficiently as did GFP fused to full-length Doa4 (Doa4-GFP; Figure 1B). Both Doa4 -GFP and Doa4-GFP were observed in cells in which the VPS4 gene had been deleted (vps4D). VPS4 encodes an ATPase that catalyzes dissociation of Doa4 (and class E Vps proteins) from endosomal membranes. Thus, the absence of Vps4 facilitates identification of Doa4 recruitment by trapping it at endosomes (Luhtala and Odorizzi, 2004). Unlike Doa4 -GFP and Doa4-GFP, GFP was predominantly cytosolic in vps4D cells when fused to amino acids 561–926 of CAT Doa4, which comprise its catalytic domain (Doa4 -GFP; Figure 1C). The N terminus of Doa4, therefore, is both necessary and sufficient for endosomal recruitment. As in the case of full-length Doa4 (Luhtala and Odorizzi, 2004), localization of Doa4 -GFP to endosomes was dependent upon Bro1 (Figure 1D), and this region co-immunoprecipi- tated with Bro1 from yeast cell lysates (Figure 1E). Figure 1 The N terminus of Doa4 is necessary and sufficient for The catalytic domain of Doa4 is specifically required Bro1-mediated localization to endosomes. (A–D) Fluorescence and for cargo deubiquitination and sorting N CAT DIC microscopy of Doa4 -GFP, Doa4-GFP, and Doa4 -GFP. FM4- To determine whether endosomal localization is sufficient to 64 is a lipophilic stain that specifically labels E compartments and confer substrate specificity, we tested if the catalytic domain vacuolar membranes. Arrowheads indicate class E compartments that do (closed arrowheads) or do not (open arrowheads) colocalize of a different UBP was functional in the MVB pathway when with GFP. Scale bar, 2.5mm. (E) Native anti-HA immunoprecipita- fused to the Doa4 N terminus. Ubp5 is the UBP in yeast most tion followed by anti-HA or anti-Bro1 immunoblotting of total lysate closely related to Doa4 (Figure 2A), but GFP fused to full- versus bound proteins. The GFP fusions in (A–D) and the HA fusion length Ubp5 (Ubp5-GFP) failed to localize to endosomes in (E) were expressed from single copies integrated into the genome under the control of the endogenous DOA4 promoter. (Figure 2B), consistent with Ubp5 having no functional role in MVB sorting. As expected, appending the Doa4 N-terminal region to the Ubp5 catalytic domain led to endosomal loca- N CAT lization of the resulting fusion protein (Doa4 -Ubp5 -GFP; activity despite being recruited to the site of Doa4 function at endosomal membranes. Figure 2C). However, GFP-tagged carboxypeptidase S (GFP- CPS), a cargo protein normally sorted into the vacuole lumen via the MVB pathway (Odorizzi et al, 1998), was missorted to A YPxL motif in the catalytic domain of Doa4 mediates N CAT the vacuole membrane in cells expressing Doa4 -Ubp5 in binding to Bro1 place of wild-type Doa4, similar to the mislocalization of The specific requirement for the Doa4 catalytic domain in the C571S GFP-CPS in doa4 cells (Figure 2D), which express a MVB pathway led us to compare its amino-acid sequence catalytically inactive Doa4 mutant. In addition, the Doa4 - with that of the Ubp5 catalytic domain. Although the two CAT Ubp5 chimera was unable to deubiquitinate CPS in vivo sequences are B60% identical, Doa4 contains a YPFL se- (Figure 2E). Altogether, these observations indicate that the quence (amino acids 826–829) versus YPYS at the corre- Ubp5 catalytic domain cannot substitute for Doa4 enzymatic sponding position in Ubp5 (Figure 2A). The YPFL in Doa4 &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 10 2007 2455 | | Doa4-mediated deubiquitination at endosomes C Richter et al Figure 2 MVB cargo sorting and deubiquitination specifically require the Doa4 catalytic domain. (A) Schematic representations of Doa4, N CAT Ubp5, and the Doa4 -Ubp5 chimera. Amino-acid identity between Doa4 and Ubp5 is indicated. Fluorescence and DIC microscopy of Ubp5- N CAT GFP (B) or Doa4 -Ubp5 -GFP (C). Arrowheads indicate class E compartments that do (closed arrowheads) or do not (open arrowheads) colocalize with GFP. (D) Fluorescence and DIC microscopy of GFP-CPS. Scale bar, 2.5 mm (B–D). (E) Denatured anti-CPS immunoprecipitations followed by anti-Ub or anti-CPS immunoblotting. Doublet bands represent differentially glycosylated forms of CPS. The GFP fusions in (B, C) were expressed from single copies integrated into the genome under the control of the endogenous DOA4 promoter. matches the consensus sequence in viral proteins, YPxL, Alix binds directly to the YPxL consensus sequence in Gag which binds Alix, the mammalian ortholog of Bro1 (Martin- protein subunits of HIV-1, EIAV, and MuLV (Martin-Serrano Serrano et al, 2003; Strack et al, 2003; Segura-Morales et al, et al, 2003; Strack et al, 2003; Segura-Morales et al, 2005). 2005). Indeed, the YPFL sequence was required for Doa4 Therefore, we used a yeast two-hybrid assay to test whether function in the MVB pathway. When this motif was changed Bro1 binds the YPFL motif in Doa4. As a positive control, we AAFA to AAFA by site-directed mutagenesis (doa4 ), deubiqui- used Snf7, which binds directly to the ‘Bro1 domain’ of Bro1 tination of CPS was defective (Figure 3B), and GFP-CPS was (Figure 4A) (Kim et al, 2005). Accordingly, Snf7 interacted mislocalized to the vacuole membrane (Figure 3A). Pulse– with both full-length Bro1 and the Bro1 domain (amino acids chase metabolic labeling followed by anti-GFP immuno- 2–387) but not with the remaining C-terminal fragment of precipitation confirmed that the cleavage of GFP from CPS, Bro1 (amino acids 388–844; Figure 4B). The catalytic domain which occurs following delivery of MVB vesicles into the of Doa4 also bound full-length Bro1 but did not interact with vacuole lumen and accurately reflects the proteolytic matura- the Bro1 domain. Instead, the Doa4 catalytic domain bound 388–844 tion of native CPS (Odorizzi et al, 1998), was quantitatively Bro1 (Figure 4B). Because the Doa4 N terminus was C571S AAFA reduced to a similar extent in doa4 and doa4 cells removed to preclude the possibility of Bro1 binding it in the compared with cleavage of GFP-CPS in wild-type cells two-hybrid assay, Bro1 interacts with the Doa4 catalytic (Figure 3C). The extent of cleavage was not significantly domain independent of the endosomal localization region C571S AAFA 388–844 different in doa4 and doa4 cells expressing lower of Doa4. Binding of Bro1 to the Doa4 catalytic domain levels of GFP-CPS (Supplementary Figure S2), nor was the was abolished, however, when the YPFL motif in Doa4 was defect in cleavage rescued upon increased Ub expression changed to AAFL (Figure 4B). Moreover, the Doa4 YPFL motif (Supplementary Figure S3), which agrees with recent work conforms to the YPxL consensus sequence because individual showing that the depleted pools of free Ub, characteristic of mutation of Y ,P ,orL , but not F , disrupted the 826 827 829 828 388–844 doa4 mutant cells, do not account for MVB sorting defects in interaction with Bro1 (Figure 4C). In addition, V and cells lacking Doa4 function (Nikko and Andre, 2007). L in Doa4 are required for this interaction, whereas I is 830 825 C571S AAFA However, GFP-CPS cleavage in doa4 and doa4 cells dispensable. was not completely blocked, as was observed in pep4D prb1D cells lacking vacuolar protease activity (Figure 3C). Thus, the Truncation of Bro1 disables it from binding the Doa4 efficiency of GFP-CPS sorting is reduced in the absence of catalytic domain and results in Doa4-specific MVB Doa4 function but not completely defective, as seen pre- pathway defects viously in cells lacking the components of the core MVB To map the YPxL-interacting region, we screened a collection sorting machinery of class E Vps proteins, including Vps4 of mutant bro1 alleles for their ability to phenocopy the MVB (vps4D; Figure 3C) (Reggiori and Pelham, 2001; Babst et al, pathway defect caused by loss of Doa4 function. A hallmark 2002). The YPFL motif was not required for recruitment of phenotype caused by deletion of the DOA4 gene is the Doa4 to endosomes (Figure 3D), indicating that, like the selective block in sorting of Ub-dependent MVB cargoes N CAT AAFA Doa4 -Ubp5 chimera (Figure 2), Doa4 is unable to such as CPS, whereas sorting of Ub-independent cargoes function in the MVB pathway despite being recruited to the such as Sna3 is unaffected (Reggiori and Pelham, 2001). In site of Doa4 activity on endosomal membranes. contrast, deletion of any class E VPS gene, including BRO1, 2456 The EMBO Journal VOL 26 NO 10 2007 &2007 European Molecular Biology Organization | | Doa4-mediated deubiquitination at endosomes C Richter et al Figure 3 The Doa4 YPxL motif is necessary for cargo sorting and deubiquitination. (A) Fluorescence and DIC microscopy of GFP-CPS. (B) Denatured anti-CPS immunoprecipitations followed by anti-Ub and anti-CPS immunoblotting. Doublet bands represent differentially glycosylated forms of CPS. The slower migrating bands in the anti-Ub panel might represent more extensive Ub modification of CPS. (C) Quantification of GFP-CPS cleavage measured after anti-GFP immunoprecipitation from S-labeled cell extracts; P-valueso0.05 (*), 0.01 (**), AAFA or 0.001 (***). (D) Fluorescence and DIC microscopy of vps4D cells expressing the Doa4 -GFP fusion from a low-copy number plasmid under the control of the DOA4 promoter. Closed arrowheads indicate class E compartments that colocalize with GFP. Scale bar, 2.5 mm (A, D). blocks sorting of both Ub-dependent and -independent car- ability of Bro1 to recruit Doa4 to endosomes (Figure 5B). goes via the MVB pathway (Figure 5A) (Reggiori and Pelham, Together, these observations suggested that truncation of 2001). Although the general mechanism by which Bro1 the Bro1 C terminus prevents it from binding the YPxL function is required for sorting MVB cargoes is unknown, motif of Doa4. we reasoned that its interaction with Doa4 constitutes a The Doa4-specific MVB sorting defect caused by the bro1-2 subdivision of function specifically required for sorting mutation was further supported by three-dimensional elec- Ub-dependent MVB cargoes. Thus, a mutation in Bro1 that tron tomographic modeling. In wild-type yeast cells, MVBs prevents it from binding the YPxL motif of Doa4 but are spherical structures that have numerous lumenal vesicles otherwise has no adverse effect on Bro1 function should (Figure 5C and Supplementary Video S5). In contrast, dele- block the sorting of CPS but not Sna3. Indeed, the bro1-2 tion of the BRO1 gene dramatically alters the morphology of allele, which contains a premature stop codon in place of endosomes such that they consist of flattened cisternae-like codon 820 (Figure 4A), caused mislocalization of GFP-CPS structures within which lumenal vesicles are entirely absent to the vacuole membrane, but did not block sorting of (Figure 5D and Supplementary Video S6). This abnormal Sna3-GFP into the vacuole lumen (Figure 5A). Furthermore, endosomal morphology is a unique characteristic caused by bro1-2 impaired GFP-CPS cleavage to a similar extent as deletion of any class E VPS gene. Therefore, these aberrant AAFA observed in doa4 cells (Figure 3C and Supplementary structures are referred to as ‘class E compartments’ Figure S2). Yeast two-hybrid analysis confirmed that (Raymond et al, 1992; Rieder et al, 1996; Babst et al, 1997; the mutant Bro1-2 protein was unable to bind the Doa4 Odorizzi et al, 1998). Unlike bro1D cells, bro1-2 cells do not catalytic domain (Figure 4B), and fluorescence microscopy have class E compartments, but instead have spherical multi- indicated that the bro1-2 mutation had no effect on the vesicular endosomes (Figure 5E and Supplementary Video &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 10 2007 2457 | | Doa4-mediated deubiquitination at endosomes C Richter et al Figure 4 Bro1 interacts with the Doa4 YPxL motif. (A) Schematic diagram of Bro1 indicating the ‘Bro1 domain’, coiled-coil (CC), and proline- rich domain (PRD). The bro1-2 mutation replaces the glutamine at position 820 with a termination codon. (B, C) Yeast two-hybrid analysis of the interaction between Doa4 and Bro1. Bro1 fragments fused to the Gal4 activation domain are indicated with GAD-Bro1, including the CAT appropriate amino-acid residues. The LexA DNA-binding domain was fused to Snf7 from A. nidulans, Doa4 , and the indicated amino-acid CAT substitutions within Doa4 . C571S S7), similar to those observed in doa4 cells (Figure 5F sequence at the C-terminal tip of Bro1 is highly conserved and Supplementary Video S8). among its candidate orthologs in other fungal species The presence of lumenal MVB vesicles in bro1-2 and (Figure 6A). Therefore, we tested whether mutation of C571S 793–844 doa4 cells is consistent with both strains having a func- its C-terminal residues affected binding of Bro1 to CAT tional MVB pathway capable of sorting Sna3-GFP but not Doa4 . As shown in Figure 6B, this interaction was Ub-dependent cargoes such as GFP-CPS. However, closer disrupted upon substitution of four alanine residues in examination revealed another phenotype caused by the place of the PSVF sequence spanning amino acids 831–834 C571S 793–844(PSVF–AAAA) bro1-2 and doa4 mutations. The lumenal MVB vesicles of Bro1 (Bro1 ), or upon individual substitu- of both mutant strains were predominantly smaller than tion of alanine for P ,S ,V ,orF . Binding also did 831 832 833 834 MVB vesicles in wild-type cells (Figure 5G) even though the not occur upon mutation of R ,M ,Y ,orY , whereas 830 838 839 842 size of MVBs was unchanged (Supplementary Figure S4B). the interaction was unaffected by substitution of alanine in Although the molecular basis for larger lumenal MVB vesicles place of D ,E ,N ,S ,K ,S ,orS (Figure 6A 835 836 837 840 841 843 844 C571S being absent in bro1-2 and doa4 cells is not clear, this and data not shown). Consistent with this region of Bro1 observation provided further evidence that truncation of Bro1 binding to the YPxL motif in Doa4, cells expressing the PSVF–AAAA mimics the MVB pathway defects caused by loss of Doa4 bro1 allele were defective in deubiquitination and function. sorting of CPS (Figure 6C and D) and contained a predomi- Overexpression of Ub restored the formation of larger nance of small lumenal MVB vesicles (Supplementary Figure lumenal vesicles in cells lacking Doa4 activity (Supplemen- S4A and Supplementary Video S9). Thus, binding of the YPxL tary Figure S3C), but also caused distortions of the limiting motif in Doa4 by the conserved C terminus of Bro1 is required endosomal membrane (Supplementary Figure S3D), resulting for Doa4-mediated deubiquitination of MVB cargoes. in structures we recently characterized as vesicular tubular endosomes (VTEs) (Nickerson et al, 2006). VTEs exist in cells lacking expression of Did2, an adaptor protein that couples Bro1 stimulates deubiquitination by Doa4 Vps4 activity to ESCRT-III dissociation from endosomes. For To test directly the effect of Bro1 on deubiquitination, we CAT unknown reasons, the lumenal vesicles of VTEs in did2D isolated recombinant Doa4 from bacteria and analyzed its cells are unusually large (Nickerson et al, 2006). Thus, it is enzymatic activity in vitro in the presence or absence of 388–844 CAT unclear whether the increased lumenal vesicle size caused by recombinant Bro1 . Incubation of GST-Doa4 with overexpression of Ub in cells lacking Doa4 function is simply Ub fused to 7-amino-4-methylcoumarin (Ub-AMC) resulted due to replenished pools of free cellular Ub or, instead, a in a low rate of hydrolysis, as measured by emission of AMC 388–844 manifestation of the VTE morphology. fluorescence (Figure 7A). Addition of His -Bro1 to the reaction dramatically enhanced the rate of hydrolysis, but A conserved C-terminal motif in Bro1 is required for its this stimulatory effect was lost when the PSVF sequence in interaction with the catalytic domain of Doa4 Bro1 was mutated (Figure 7A), confirming that this motif is Based on the inability of the Bro1-2 protein to bind the Doa4 essential for binding to the Doa4 catalytic domain and catalytic domain, we hypothesized that a C-terminal fragment stimulation of deubiquitination. Maximal stimulation of 388–844 of Bro1 would be sufficient for this interaction to occur. Ub-AMC hydrolysis was reached when His -Bro1 was CAT Indeed, the last 52 amino acids of Bro1 (residues 793–844) present at 1- to 2-fold amounts relative to GST-Doa4 CAT interacted with Doa4 (Figure 4B). The amino-acid (Figure 7B), indicating a stoichiometric relationship. 2458 The EMBO Journal VOL 26 NO 10 2007 &2007 European Molecular Biology Organization | | Doa4-mediated deubiquitination at endosomes C Richter et al Figure 5 The C terminus of Bro1 is specifically required for Ub-dependent cargo sorting. (A) Fluorescence and DIC microscopy of GFP-CPS and Sna3-GFP. (B) Fluorescence and DIC microscopy of Doa4-GFP. Arrowheads indicate E compartments that do (closed arrowheads) or do not C571S (open arrowheads) colocalize with GFP. Scale bar, 2.5mm (A, B). (C–F) EM showing MVBs in wild-type, bro1-2, and doa4 cells, and the class E compartment in bro1D cells. Vesicles in tomographic models have diameters indicated by green (18–23 nm), purple (24–29 nm), pink (30–35 nm), red (36–47 nm). V¼ vacuole. Scale bar, 0.1mm(G) Distribution of MVB vesicle diameters from wild-type (n¼ 330), bro1-2 C571S C571S (n¼ 265), and doa4 cells (n¼ 371). Mean vesicle diameters of bro1-2 and doa4 cells differ significantly from that of wild-type cells (Po0.0001). 388–844 To determine whether Bro1 increases the catalytic Discussion activity and/or substrate affinity of Doa4, we generated Michaelis–Menten curves by testing the reaction velocity at Bimodal regulation of localization and activation is emerging varying Ub-AMC concentrations. As shown in Figure 7C, as a paradigm for controlling the specificity of enzymes that CAT 388–844 GST-Doa4 incubated with His -Bro1 yielded a V function in multiple cellular processes (Bhattacharyya et al, 6 max of Ub-AMC hydrolysis at 75.16 nmole/min, resulting in a K 2006; Chen and Kass, 2006; Dard and Peter, 2006). Doa4 is a CAT of 0.56 mM. However, the reaction velocity for GST-Doa4 UBP in S. cerevisiae that removes Ub from membrane pro- alone was linearly related to substrate concentration teins targeted into the MVB pathway (Dupre and (Figure 7C), which precluded determination of the V and Haguenauer-Tsapis, 2001; Katzmann et al, 2001; Losko max K . Nevertheless, we infer from Figure 7C that the K of et al, 2001), and from soluble proteins targeted to protea- M M CAT Ub-AMC hydrolysis by GST-Doa4 alone is significantly somes (Papa et al, 1999). In addition, Doa4 activity has been CAT higher than the K of GST-Doa4 in the presence of His - implicated in the coordination of DNA replication (Singer M 6 388–844 Bro1 , suggesting that Bro1 increases the affinity of et al, 1996) and the DNA damage response (Fiorani et al, Doa4 for its substrate. 2004). Our results indicate that Bro1 both recruits Doa4 to &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 10 2007 2459 | | Doa4-mediated deubiquitination at endosomes C Richter et al Figure 6 The Bro1 PSVF motif interacts with the Doa4 YPxL motif. (A) Multiple sequence alignment of the C termini of Bro1 homologs from several fungal species. Arrowheads indicate Bro1 residues CAT required for interaction with Doa4 by two hybrid. (B) Yeast two-hybrid analysis of the interaction between Doa4 and Bro1. (C) Fluorescence and DIC microscopy of GFP-CPS. Scale bar, 2.5mm. (D) Anti-CPS immunoblot of whole-cell lysates. TheB9 kDa shift in CPS corresponds to its monoubiquitinated form. late endosomes and stimulates its catalytic activity, thereby exerting dual modes of regulation to control the specificity of Doa4 function in the MVB pathway. Site-specific activation by Bro1 ensures that Doa4 is active during its transient associa- Figure 7 Bro1 stimulates Doa4-mediated deubiquitination in vitro. tion with endosomes and implies that the location and timing (A) Measurement of fluorescence generated by deubiquitination of Ub-AMC (0.4mM; l : 380 nM, l : 440 nM) using 50 nM of Doa4 activity outside of the MVB pathway is also regulated ex em 406–926 388–844 GST-Doa4 with or without 100 nM His -Bro1 or His - 6 6 to prevent nonspecific protein deubiquitination. Dual-regula- 388–844(PSVF–AAAA) Bro1 . All Bro1 and Doa4 proteins were purified tory mechanisms may similarly function in other cases of from E. coli and reactions were run for 7 min in triplicate. UBP-mediated deubiquitination. The catalytic activity of Measurements were also taken in the absence of either enzyme or substrate to control for aberrant deubiquitination or fluorescence, Ubp6 is stimulated by its recruitment to proteasomes respectively. (B) Doa4-mediated Ub-AMC hydrolysis under varying (Leggett et al, 2002), as is the activity of Ubp8 upon its 388–844 388–844(PSVF–AAAA) concentrations of His -Bro1 or His -Bro1 . 6 6 assembly into the SAGA histone acetyltransferase complex 406–926 GST-Doa4 and Ub-AMC concentrations were kept constant (Lee et al, 2005). In addition, the catalytic activity of AMSH, at 50 nM and 0.4 mM, respectively. The deubiquitination rate 406–926 was normalized to that of GST-Doa4 in the absence of which belongs to the JAMM metalloprotease family of DUBs, 388–844 406–926 His -Bro1 .(C) Ub-AMC hydrolysis by GST-Doa4 is stimulated upon binding to the endosomal sorting factor (50 nM) under varying substrate concentrations, with or without STAM (McCullough et al, 2006). 388–844 His -Bro1 (100 nM). All data points are represented by The finding that Bro1 reduces the K of deubiquitination is M mean7s.e.m. consistent with a model in which it enhances substrate binding by Doa4, although the mechanism by which this catalytic autoinhibitory C terminus (Yao et al, 2006). occurs is unknown. In mammalian cells, Adrm1 reduces the Although Uch37 belongs to the Ub C-terminal hydrolase K of Uch37-mediated deubiquitination by binding its non- (UCH) family of DUBs, structural analyses indicate that the 2460 The EMBO Journal VOL 26 NO 10 2007 &2007 European Molecular Biology Organization | | Doa4-mediated deubiquitination at endosomes C Richter et al catalytic cores of UCH and UBP enzymes are very similar in this manner by binding the YPxL motif of Doa4 was guided (Johnston et al, 1999; Hu et al, 2002). The ability of Adrm1 to by previous work showing that the mammalian ortholog of relieve autoinhibition by binding the non-catalytic C-terminal Bro1, Alix, binds a YPxL consensus sequence in Gag protein region of Uch37, however, is distinct from the mechanism by subunits encoded by HIV-1, EIAV, and MuLV. As a conse- which Bro1 binds directly to the catalytic domain of Doa4 to quence, Alix is recruited to the site of viral assembly to stimulate its activity. Recent structural analysis of UBPY, the facilitate budding of infectious virions from host cells mammalian UBP most closely related to Doa4, has revealed it (Puffer et al, 1997; Martin-Serrano et al, 2003; Strack et al, to be autoinhibited by b-strands and loop segments in its 2003; Segura-Morales et al, 2005). Like MVB cargoes, viral catalytic domain that block access to the Ub-binding site, and Gag proteins are ubiquitinated, but it is unclear whether Ub only after these elements are displaced, is UBPY capable of modification is relevant to viral budding (Morita and substrate binding and deubiquitination (Avvakumov et al, Sundquist, 2004). However, it is noteworthy that transplanta- 2006). Movement of these features in UBPY was proposed to tion of the YPxL motif into recombinant viral constructs be under the control of helix 12, which might act as a hinge. reduces the amount of Ub-conjugated Gag (Martin-Serrano Alignment of the UBPY and Doa4 catalytic domain sequences et al, 2004), suggesting that Alix subsequently results in positions the YPxL motif of Doa4 adjacent to helix 12 of UBPY recruitment of a DUB in a way similar to recruitment of (Avvakumov et al, 2006), raising the possibility that binding Doa4 by Bro1. of Bro1 to this site induces a structural rearrangement that YPxL motifs also mediate protein interactions with homo- provides the catalytic site of Doa4 access to ubiquitinated logs of Bro1 that have no functional connection to the MVB MVB cargoes. However, this interaction is likely to be tran- pathway. Rim101 in S. cerevisiae and PacC in Aspergillus sient, as we cannot detect stable binding between GST- nidulans are YPxL-containing transcription factors that un- CAT 388–844 Doa4 and His -Bro1 in vitro (C Richter and G dergo proteolytic cleavage and activation upon interacting Odorizzi, unpublished observations). with the Bro1 homologs Rim20 and PalA, respectively The role of the N terminus of Doa4 in its endosomal (Xu and Mitchell, 2001; Vincent et al, 2003). Although the recruitment is consistent with a modular principle of UBPs C-terminal PSVF-containing sequence in Bro1 is conserved in which their non-catalytic domains mediate localization to among the predicted functional orthologs of Bro1 in fungal specific subcellular sites of function (Leggett et al, 2002; Hu species, it is not found in Rim20 and PalA. Similarly, the C et al, 2005). Recent work identified four conserved amino- termini of Bro1 and Alix share little sequence homology, and acid motifs in the N-terminal region of Doa4 that are required the region of Alix that binds viral YPxL sequences is located for its localization to endosomes (Amerik et al, 2006). in the middle of the protein between its Bro1 domain and However, based on analysis of an overexpressed Doa4-GFP proline-rich region (Fisher et al, 2007; Lee et al, 2007). fusion protein, the same study concluded that Bro1 is dis- Therefore, any similarity between the PSVF motif of Bro1 pensable for endosomal localization of Doa4 (Amerik et al, and the YPxL-binding regions of Alix, Rim20, and PalA might 2006). Although Bro1 physically associates with the N-term- only be apparent at the level of tertiary structure. inal endosomal localization region of Doa4 in cell lysates Mutations that disrupt the PSVF motif in Bro1 phenocopy (this study) and is required for the endosomal localization of the loss of Doa4 function by blocking the sorting of CPS, a Doa4-GFP expressed at physiologically normal levels (this Ub-dependent MVB cargo, whereas the sorting of Sna3, a Ub- study and Luhtala and Odorizzi, 2004), we have also found independent cargo, continues normally. Accordingly, bro1-2 PSVF–AAAA C571S that, when overexpressed, Doa4-GFP localizes to endosomes and bro1 mutant cells both resemble doa4 cells in the absence of Bro1 (C Richter and G Odorizzi, unpub- in that they have MVBs rather than the class E compartments lished observations). It is likely, therefore, that another that are characteristically observed upon deletion of BRO1 or component on endosomal membranes binds Doa4, but this any other class E VPS gene (Raymond et al, 1992; Rieder et al, factor, alone, is insufficient to effectively concentrate Doa4 at 1996; Babst et al, 1997; Odorizzi et al, 1998). Defining how endosomes under the normal conditions of low Doa4 expres- class E Vps proteins coordinate both MVB cargo sorting and sion. Such a factor may also stabilize the interaction between lumenal vesicle formation is a major challenge in under- Bro1 and the Doa4 N terminus because their binding is standing the molecular mechanism of MVB function. Our undetectable in the yeast two-hybrid assay (C Richter and G results clearly indicate that a division of labor exists for Bro1 Odorizzi, unpublished observations). in these processes. Insight into the specific role that Bro1 has In addition to causing MVB sorting defects, loss of Doa4 in forming lumenal MVB vesicles might be gleaned from function renders cells hypersensitive to canavanine, a cyto- studies indicating that its mammalian ortholog, Alix, binds toxic arginine analog (Papa and Hochstrasser, 1993). The lysobisphospatidic acid (LBPA) to regulate the dynamic abil- canavanine hypersensitivity can be rescued by fusion of the N ity of this lipid to promote either fission or fusion of lumenal terminus of Doa4 to the catalytic domain of its most closely MVB membranes (Matsuo et al, 2004; Le Blanc et al, 2005). related yeast homolog, Ubp5 (Amerik et al, 2006). However, However, a similar role for Bro1 in regulating membrane we found that MVB cargo deubiquitination and sorting could dynamics likely differs mechanistically because LBPA has not not be rescued by an identical Doa4-Ubp5 fusion protein (nor been detected in yeast. could GFP-CPS cleavage; C Richter and G Odorizzi, unpub- The range of MVB vesicle diameters observed in wild-type lished observation), indicating that canavanine sensitivity is yeast cells sharply contrasts with the predominantly small caused by a defect in Doa4 function unrelated to the MVB vesicles observed in cells lacking Doa4 activity. How lumenal pathway. The inability of the Ubp5 catalytic domain to MVB vesicle size correlates with Doa4 function is unknown. substitute for that of Doa4 led us to speculate that an If deubiquitination has a direct role in MVB vesicle size endosome-associated factor might function specifically to determination, it might be coupled to activation of a mole- stimulate Doa4 activity. Our discovery that Bro1 functions cular machinery that controls the formation of larger vesicles &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 10 2007 2461 | | Doa4-mediated deubiquitination at endosomes C Richter et al (Odorizzi et al, 2003) and mouse anti-HA monoclonal antibodies within which Ub-dependent cargoes such as CPS are selec- (Covance). For pulse–chase metabolic labeling, 5 A equivalents tively packaged, while a separate machinery might operate of yeast cells were incubated with 100 mCi S-labeled methionine/ independently to form smaller vesicles that transport cysteine for 10 min at 301C, followed by the addition of chase Ub-independent cargoes such as Sna3. Alternatively, lumenal mixture (5 mM methionine, 1 mM cysteine, and 0.2% yeast extract) for 0 or 30 min. Afterward, cells were precipitated by the addition of MVB vesicle size might be determined by the influx of 10% (vol/vol) trichloroacetic acid, cell lysates were prepared under cargoes into the pathway. Most yeast cargoes are dependent denaturing conditions, and immunoprecipitations of GFP-CPS were upon the cycle of ubiquitination and deubiquitination and, as performed as previously described (Katzmann et al, 2001; Luhtala such, are blocked from entering the MVB pathway in cells and Odorizzi, 2004) using anti-GFP polyclonal antiserum (Odorizzi et al, 1998). Immunoprecipitates were resolved by SDS–PAGE and lacking Doa4 activity (Loayza and Michaelis, 1998; Dupre exposed to a storage phosphor screen (GE Healthcare), and then and Haguenauer-Tsapis, 2001; Reggiori and Pelham, 2001; developed by a Storm Phosphorimager (GE Healthcare) and Urbanowski and Piper, 2001). Deubiquitination might, there- TM quantified using ImageQuant TL (GE Healthcare). All samples fore, have no direct influence on MVB vesicle formation, with were tested in triplicate and the Prism software (Graphpad Software) was used for statistical analysis. smaller vesicles providing adequate surface area to accom- modate a decreased cargo load in the absence of Doa4 Isolation of the bro1-2 allele function. BRO1 was PCR-amplified using Taq polymerase (Invitrogen) under error-prone conditions (Guthrie and Fink, 2002) to generate a library of random mutant bro1 alleles that were pooled and Materials and methods cotransformed into yeast strain KGY1 (Luhtala and Odorizzi, 2004) together with pGO221 (pRS416 containing the BRO1 gene) Yeast strains and plasmid construction that had been linearized by EcoRI/HindIII digestion. In vivo For information, see Supplementary data. homologous recombination yielded 410 000 colonies of KGY1, each of which contained one repaired plasmid encoding a single Fluorescence microscopy bro1 allele. KGY1 expresses the carboxypeptidase Y-invertase Strains were grown to logarithmic phase at 301C in synthetic reporter fusion protein, which is secreted upon loss of Bro1 medium before observation at room temperature using a Zeiss function (Luhtala and Odorizzi, 2004). Thus, colonies of KGY1 Axioplan 2 microscope with an NA 1.40 oil-immersion objective transformed with plasmids encoding non-functional mutant bro1 (Carl Zeiss MicroImaging Inc.). Fluorescence and differential alleles were identified based on the detection of secreted invertase interference contrast (DIC) images were acquired with a Cooke activity using a colorimetric agar overlay assay (Darsow et al, 2000; Sensicam digital camera (Applied Scientific Instruments Inc.) and Luhtala and Odorizzi, 2004). Whole-cell lysates prepared from processed using Slidebook (Intelligent Imaging Innovations) and clones secreting invertase were analyzed by SDS–PAGE and Western Photoshop 7.0 software (Adobe). Cells were stained with FM4-64 blotting using rabbit anti-Bro1 antiserum to eliminate plasmids not (Molecular Probes Inc.) using a pulse–chase procedure as described expressing Bro1. The remaining plasmids were isolated from yeast previously (Odorizzi et al, 2003). and subjected to DNA sequence analysis. Among the mutant bro1 alleles isolated by this procedure, the bro1-2 allele was the only one High-pressure freeze substitution and electron tomography that was capable of sorting Sna3-GFP but not GFP-CPS via the MVB Cells were high-pressure frozen, freeze-substituted with 0.1% pathway. uranyl acetate, 0.25% glutaraldehyde, anhydrous acetone at 901C, embedded in Lowicryl HM20, and polymerized under UV Yeast two-hybrid at 501C (Winey et al, 1995). Semi-thick sections (200 nm) were CTY10.5d was transformed with GAD and LEXA fusions (Vincent placed on rhodium-plated formvar-coated copper slot grids and et al, 2003), patched onto agar medium, allowed to grow for 3 days, mapped on a Phillips CM10 TEM at 80 kV. Dual tilt series images then lysed by exposure to chloroform vapor for 30 min before were collected from þ 601 to 601 with 11 increments at 200 kV overlay with 0.6% agar containing Z-buffer (60 mM Na HPO , 2 4 using a Tecnai 20 FEG (FEI). Tomograms were imaged at  29 000 . . 40 mM NaH PO H 0, 10 mM KCl, 1 mM MgSO 7H 0, 0.2% 2 4 2 4 2 with a 0.77 nm pixel (binning 2). Sections were coated on both sides b-mercaptoethanol, and 0.67 mg/ml X-gal). Blue/white analysis with 15-nm fiducial gold for reconstruction of back projections was performed after 6 h to identify interactions. using IMOD software (Kremer et al, 1996). 3dmod software was used for mapping structure surface areas. Mean z-scale values for In vitro deubiquitination assays sections were within 3%. Best-fit sphere models were used to 406926 388–844 388–844(PSVF–AAAA) GST-Doa4 , His -Bro1 , and His -Bro1 6 6 measure vesicle diameters to the outer leaflet of membrane bilayers. were expressed in Escherichia coli BL21-CodonPlus (DE3) cells IMOD calculated limiting membrane surface areas using three- (Stratagene) by induction with 0.5 mM isopropyl-b-D-thiogalacto- dimensional mesh structures derived from closed contours drawn side at 201C for 18 h and purified using glutathione–Sepharose (GE each 3.85 nm using imodmesh. For quantitation, vesicles from 12– Healthcare) or TALON metal affinity resin (Clontech). For deubi- 13 random MVBs were measured from 4–5 cells of each strain and 406–926 quitination reactions, 50 nM GST-Doa4 was incubated with or analyzed using the Gaussian curve fit of the Prism software 388–844 388–844(PSVF–AAAA) without His -Bro1 , or His -Bro1 , in reaction 6 6 (GraphPad Software). buffer (50 mM HEPES pH 7.5, 2 mM DTT, and 0.1 mg/ml BSA) for 30 min at 251C in 96-well plates. Reactions were initiated by the Immunoprecipitation and Western blotting addition of Ub-AMC (Boston Biochem) and analyzed by a Tecan Denatured immunoprecipitations to detect Ub-CPS were performed Safire II fluorescence plate reader (l : 380 nM, l : 440 nM), which ex em as described previously (Katzmann et al, 2001; Luhtala and maintained the reaction at 251C. All samples were tested in Odorizzi, 2004). The 0.5 A equivalents were resolved by SDS– 600 triplicate and Prism (GraphPad Software) was used for statistical PAGE, transferred to nitrocellulose, and analyzed by Western blot analysis and nonlinear fit to Michaelis–Menten kinetics. using rabbit anti-CPS polyclonal antiserum (Cowles et al, 1997), anti-Ub monoclonal antibodies (Zymed), and anti-phosphoglyce- Supplementary data rate kinase (PGK) monoclonal antibodies (Invitrogen). For native Supplementary data are available at The EMBO Journal Online 1–560 immunoprecipitations of Doa4 -TEV-HA, yeast lysates prepared (http://www.embojournal.org). as previously described (Luhtala and Odorizzi, 2004) were incubated with mouse anti-HA monoclonal antibodies (Covance) and protein G–Sepharose beads (GE Healthcare) for 2 h at 41C, after Acknowledgements which the beads were collected by centrifugation and washed (Luhtala and Odorizzi, 2004), and 10 A equivalents of We thank Sarah Altschuler and Megan Wemmer (University of immunoprecipitates or 0.5 A equivalents of total lysate were Colorado) for plasmid and yeast strain construction, Doug Burch resolved by SDS–PAGE, transferred to nitrocellulose, and analyzed (University of Colorado) for yeast strain construction and electron by Western blot using rabbit anti-Bro1 polyclonal antiserum microscopy, Amy Palmer (University of Colorado) for the use of the 2462 The EMBO Journal VOL 26 NO 10 2007 &2007 European Molecular Biology Organization | | Doa4-mediated deubiquitination at endosomes C Richter et al Tecan Safire II fluorescence plate reader, Olivier Vincent (Centro de and Eric Cooper (Johns Hopkins University) for advice on DUB Investigaciones Biologicas del CSIC) for providing the yeast two- kinetic analysis. 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Journal

The EMBO Journal – Springer Journals

Published: May 16, 2007

Keywords: deubiquitination; endosomes; multivesicular bodies; protein sorting

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