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Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes

Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes JCB Article Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes Natalie Luhtala and Greg Odorizzi Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 biquitination directs the sorting of cell surface recep- of DOA4 restores cargo protein deubiquitination and sorting via the MVB pathway and reverses the abnormal endo- tors and other integral membrane proteins into the multivesicular body (MVB) pathway. Cargo proteins somal morphology typical of bro1 mutant cells, resulting in the restoration of multivesicular endosomes. We further are subsequently deubiquitinated before their enclosure within MVB vesicles. In Saccharomyces cerevisiae, Bro1 demonstrate that Doa4 interacts with Bro1 on endosomal membranes and that the recruitment of Doa4 to endosomes functions at a late step of MVB sorting and is required for cargo protein deubiquitination. We show that the loss of requires Bro1. Thus, our results point to a key role for Bro1 in coordinating the timing and location of deubiquitination Bro1 function is suppressed by the overexpression of DOA4, which encodes the ubiquitin thiolesterase required for the by Doa4 in the MVB pathway. removal of ubiquitin from MVB cargoes. Overexpression Introduction Multivesicular bodies (MVBs) are late endosomes contain- the linkage of a single Ub subunit (or a chain of two to three ing lumenal vesicles formed by invagination of the limiting subunits) to their cytoplasmic domains (Hicke and Dunn, endosomal membrane. The MVB vesicles are delivered into 2003). Ub is removed from both polyubiquitinated and the hydrolytic lumen of the lysosome/vacuole upon fusion monoubiquitinated proteins by deubiquitinating enzymes, of the limiting MVB membrane with the lysosomal/vacuolar thereby enabling cells to maintain a constant pool of Ub membrane. A variety of cell surface receptors down-regulated (Weissman, 2001; Wing, 2003). from the plasma membrane are sorted into MVB vesicles en The sorting of ubiquitinated MVB cargoes is controlled route to being degraded, including many growth factor re- by class E Vps proteins, a set of conserved cytoplasmic pro- ceptor tyrosine kinases in animals and G protein-coupled teins that associate with endosomal membranes. Several class pheromone receptors in the budding yeast Saccharomyces E Vps proteins coassemble into distinct complexes that bind cerevisiae. In yeast, several biosynthetic enzymes are also sorted to ubiquitinated cargo proteins and guide them into the into MVB vesicles during their transport from the Golgi to MVB pathway. In yeast, cargoes initially bind the Vps27– the vacuole (for review see Hicke and Dunn, 2003). Hse1 complex and subsequently interact with the ESCRT-I Ubiquitination mediates the sorting of integral membrane complex (Vps23, Vps28, and Vps37; Bilodeau et al., 2003; proteins into the MVB pathway. Ubiquitin (Ub) is a highly Katzmann et al., 2003). The ESCRT-II (Vps22, Vps25, and conserved 76-aa polypeptide that is covalently linked to specific Vps36) and ESCRT-III (Vps2, Vps20, Vps24, and Snf7) protein substrates by a cascade of Ub-conjugation enzymes. complexes function downstream of ESCRT-I (Babst et al., Ubiquitination was first characterized to occur on soluble 2002a, b), but their precise roles are not known. In mamma- proteins that are polyubiquitinated via the attachment of a lian cells, class E Vps orthologues also mediate the sorting of chain of four or more Ub subunits, which targets these sub- MVB cargo proteins (Hicke and Dunn, 2003). Further- strates for degradation by the proteasome (Weissman, 2001). more, this machinery is exploited by certain enveloped viruses In contrast, MVB cargo proteins are monoubiquitinated via in order to escape from host cells. For example, Tsg101, the mammalian orthologue of yeast Vps23, is recruited to HIV-1 budding sites at the plasma membrane by directly interacting The online version of this article contains supplemental material. Address correspondence to Greg Odorizzi, Molecular, Cellular, and Developmental Biology, 347 UCB, University of Colorado, Boulder, Abbreviations used in this paper: CPS, carboxypeptidase S; CPY, carboxy- CO 80309-0347. Tel.: (303) 735-0179. Fax: (303) 492-7744. email: peptidase Y; CPY-Inv, CPY-invertase; DIC, differential interference con- [email protected] trast; MVB, multivesicular body; Ub, ubiquitin; UBP, ubiquitin-specific Key words: endosomes; protein sorting; ubiquitin thiolesterase processing protease. © The Rockefeller University Press, 0021-9525/2004/08/717/13 $8.00 The Journal of Cell Biology, Volume 166, Number 5, August 30, 2004 717–729 http://www.jcb.org/cgi/doi/10.1083/jcb.200403139 717 The Journal of Cell Biology 718 The Journal of Cell Biology | Volume 166, Number 5, 2004 with ubiquitinated viral Gag proteins (for review see Pornillos et al., 2002). Additional class E Vps proteins function in the MVB pathway in yeast, including Vps4, an AAA-type ATPase that catalyzes the dissociation of ESCRT complexes from endo- somal membranes (Babst et al., 2002a,b). Vps4 also func- tions in the endosomal dissociation of Bro1, a class E Vps protein required for the deubiquitination of cargo proteins at a late stage of the MVB pathway downstream of the ES- CRT-III complex (Nikko et al., 2003; Odorizzi et al., 2003). The association of Bro1 with endosomes requires Snf7 (Odorizzi et al., 2003), and the mammalian Bro1 ortho- logue, Alix (also known as Aip1), has been shown to bind the mammalian Snf7 orthologue, CHMP4b (Katoh et al., 2003; Martin-Serrano et al., 2003; Peck et al., 2004; Strack et al., 2003; von Schwedler et al., 2003). Studies of HIV-1– infected cells have revealed that Alix/Aip1 also binds Tsg101 during viral budding from the plasma membrane (Strack et al., 2003; von Schwedler et al., 2003), suggesting that Alix/ Figure 1. The coiled-coil domain of Bro1 is essential for CPS transport via the MVB pathway. (A) Schematic diagram of Bro1 Aip1 bridges an interaction between the ESCRT-I and -III indicating the locations of the Bro1 domain (BOD), the coiled-coil complexes. Interestingly, Alix/Aip1 binds specifically to syn- domain (CC), and the proline-rich domain (PRD). (B and C) Fluores- thetic liposomes containing lysobisphosphatidic acid and ap- cence and DIC microscopy of BHY10, GOY57 and KGY1 cells pears to antagonize the ability of this unconventional phos- expressing a GFP-CPS fusion. In C, cells were transformed with a CC pholipid to promote membrane invagination (Matsuo et al., high-copy (2 ) plasmid encoding bro1 or wild-type BRO1. Arrowheads indicate class E compartments (B) and class E com- 2004), but the role of Alix/Aip1 in controlling membrane partment-like structures (C). Bars, 2.5 m. dynamics is not clear. Here, we report that the defects in MVB sorting caused by the loss of Bro1 function in yeast are suppressed by over- expression of the DOA4 gene, which encodes the Ub thio- nome in place of the wild-type BRO1 gene. To determine CC lesterase required for the removal of Ub from MVB cargo whether MVB sorting was functional in bro1 cells, we ex- proteins (Dupre and Haguenauer-Tsapis, 2001; Katzmann amined the intracellular localization of a fusion protein in et al., 2001; Losko et al., 2001). Specifically, our data dem- which GFP was attached to the cytoplasmic domain of car- onstrate that high-copy expression of DOA4 restores cargo boxypeptidase S (CPS), a biosynthetic enzyme that is sorted protein deubiquitination and subsequent transport via the via the MVB pathway during its transport from the Golgi to MVB pathway in bro1 mutant cells. The catalytic activity the vacuole (Odorizzi et al., 1998). GFP-CPS was found en- of Doa4 is essential for its role as a suppressor, indicating tirely within the vacuole lumen of wild-type cells (Fig. 1 B). CC that the mechanism of suppression involves substrate deu- In contrast, GFP-CPS in bro1 cells was observed at the biquitination. Furthermore, the multivesicular morphology vacuole membrane (Fig. 1 B), which is indicative of a defect of late endosomes, which is abnormal in bro1 mutant cells, in sorting of the fusion protein via the MVB pathway is restored by the overexpression of DOA4. The ability of (Odorizzi et al., 1998). GFP-CPS was also mislocalized to high-copy DOA4 to suppress the loss of Bro1 function is class E compartments (Fig. 1 B, arrowheads), abnormal late explained by our finding that Bro1 is essential for the local- endosomal structures that occur in all class E vps mutant ization of Doa4 to endosomes and that Bro1 and Doa4 cells. A similar defect in the localization of GFP-CPS was physically interact on endosomal membranes. Therefore, seen in bro1 cells in which the entire BRO1 coding se- Bro1 has a crucial role in coordinating substrate deubiquiti- quence had been deleted (Fig. 1 B). In addition, we observed CC nation in the MVB pathway by recruiting Doa4 to endo- in both bro1 and bro1 mutant cells that Ste3-GFP, an somes. MVB cargo protein endocytosed from the plasma mem- brane (Urbanowski and Piper, 2001), failed to be sorted into the vacuole lumen (unpublished data). Thus, the coiled-coil domain of Bro1 is essential for the function of Bro1 in the Results MVB pathway. CC The coiled-coil domain of Bro1 is essential for Interestingly, when we overexpressed the bro1 mutant MVB sorting allele in wild-type cells, GFP-CPS was mislocalized both to Many class E Vps proteins have one or more coiled-coil do- the vacuole membrane and to a structure that resembles the mains, which are structural elements that typically mediate class E compartment (Fig. 1 C). In contrast, overexpression protein–protein interactions. Bro1 has a central coiled-coil of the wild-type BRO1 gene had no deleterious effect on the domain spanning residues 543-583 (Fig. 1 A). We con- localization of GFP-CPS (Fig. 1 C). The overexpression of CC CC structed a mutant allele (bro1 ) in which the sequence en- bro1 , therefore, causes a dominant-negative phenotype, coding this domain had been deleted, and then integrated possibly because the mutant gene product interferes with the CC the bro1 allele by homologous recombination into the ge- function of the wild-type Bro1 protein. Bro1 recruits Doa4 to endosomes | Luhtala and Odorizzi 719 GFP, fluorescence was localized at class E compartments that were costained by FM 4-64 (Fig. 2, C and D, closed ar- rowheads). Class E compartments had formed in VPS4 CC cells expressing bro1 -GFP in place of wild-type BRO1 due to the fact that the coiled-coil domain is required for the function of Bro1 in the MVB pathway (Fig. 1). Electron mi- croscopic analysis of immunogold labeled cell sections con- CC firmed that the bro1 -GFP fusion protein was, indeed, as- sociated with class E compartments in both VPS4 and vps4 cells (not depicted). Interestingly, in contrast with CC wild-type Bro1-GFP, bro1 -GFP was also localized in both strains to additional punctate structures that were not labeled by FM 4-64 (Fig. 2, C and D, open arrowheads). CC However, the localization of bro1 -GFP to class E com- partments indicates that the coiled-coil domain per se is not required for the localization of Bro1 to late endosomes. Overexpression of DOA4 suppresses CPY sorting defects in bro1 mutant cells To identify proteins that may be able to restore normal function of the MVB pathway when over-produced in the CC bro1 mutant strain, we designed a genetic screen based upon another phenotype observed in bro1 mutant cells. In addition to MVB sorting defects, bro1 mutants inefficiently sort a soluble vacuolar enzyme, carboxypeptidase Y (CPY). Newly synthesized CPY is transported from the ER to the Golgi, where it binds a transmembrane receptor, Vps10. In wild-type cells (Fig. 3 A), the Vps10–CPY complex is sorted from the Golgi to endosomes, whereupon CPY dissociates Figure 2. The Bro1 coiled-coil domain is not required for endosomal from Vps10, and the receptor recycles back to the Golgi, localization of Bro1. Fluorescence and DIC microscopy of DMY1 whereas CPY is transported further toward the vacuole (Cere- (A), DMY2 (B), DBY15 (C), and DBY18 (D) cells stained with FM ghino et al., 1995; Cooper and Stevens, 1996). The class E 4-64. Closed arrowheads indicate class E compartments costained compartments that are formed in bro1 mutant cells prevent by FM 4-64. Open arrowheads indicate GFP-positive structures not labeled by FM 4-64. Bars, 2.5 m. Vps10 from efficiently recycling to the Golgi (Fig. 3 B), re- sulting in a significant portion of newly synthesized CPY en- tering by default into the secretory pathway (Odorizzi et al., The coiled-coil domain is not required for Bro1 to 2003). Thus, the secretion of CPY by bro1 mutant cells is associate with endosomes likely to be an indirect consequence of the aberrant endoso- We previously demonstrated that Bro1 is a cytoplasmic pro- mal morphology that occurs upon the loss of Bro1 function. tein which associates with endosomal compartments and that The percentage of total cellular CPY that is secreted can be the dissociation of Bro1 from endosomes requires the ATPase measured quantitatively using a colorimetric assay that moni- activity of Vps4 (Odorizzi et al., 2003). Thus, when we re- tors the enzymatic activity of a CPY-invertase (CPY-Inv) fu- placed the wild-type BRO1 gene with a genomically inte- sion protein (Darsow et al., 2000). As shown in Fig. 3 C, grated BRO1-GFP gene fusion in cells that have normal Vps4 wild-type cells secreted 1% of CPY-Inv, whereas bro1 CC function (VPS4 ), we observed multiple fluorescent punctate cells secreted 10–15 times this amount. The bro1 mutant structures in addition to diffuse cytoplasmic fluorescence (Fig. strain secreted nearly the same percentage as bro1 cells (Fig. 2 A). Upon deletion of the VPS4 gene (vps4), the GFP fluo- 3 C), which is consistent with the coiled-coil domain being rescence was concentrated at class E compartments, which essential for the function of Bro1 in the MVB pathway. had formed in these cells due to the absence of Vps4 activity The colorimetric assay for invertase activity can also be (Fig. 2 B). The cells in these experiments shown in Fig. 2 (and used to detect the secretion of CPY-Inv by cells growing on in Figs. 6, 7, and 9) were also labeled by a brief pulse of FM solid medium (Darsow et al., 2000). We used this assay in a 4-64 followed by incubation in label-free medium. FM 4-64 genetic screen to identify any gene that, when overexpressed CC is a fluorescent lipophilic compound that intercalates into the in bro1 cells, reduced the amount of secreted CPY-Inv. plasma membrane and is endocytosed, resulting in the stain- We reasoned that such a high-copy suppressor would encode ing of vacuole membranes and class E compartments (Vida a protein that interacts with Bro1 and would be able to re- and Emr, 1995). store normal function of the MVB pathway when over pro- CC To determine if the coiled-coil domain is required for duced. Thus, we transformed the bro1 strain with a high- the association of Bro1 with endosomes, we integrated a copy plasmid library of yeast genomic DNA and identified CC CC bro1 -GFP gene fusion in place of the wild-type BRO1 colonies that secreted less CPY-Inv relative to the bro1 CC gene. Both in VPS4 and vps4 cells expressing bro1 - strain. One clone contained a plasmid that could reproduc- 720 The Journal of Cell Biology | Volume 166, Number 5, 2004 mately twofold the amount of CPY-Inv secreted by bro1 cells, indicating a partial bypass of Bro1. The catalytic region of UBP enzymes have a conserved three-domain architecture that includes a critical cysteine residue, which forms a thiolester bond with Ub (Hu et al., C/S 2002). We constructed a point mutant allele (doa4 ) in which the corresponding cysteine in Doa4 (Cys ) had been C/S changed to a serine residue. The doa4 allele failed to suppress CPY-Inv secretion when overexpressed in either CC C/S bro1 or bro1 cells. Instead, doa4 overexpression en- hanced the mutant phenotype in both strains and caused a stronger dominant-negative phenotype in wild-type cells (Fig. 3 E). Catalytic activity, therefore, is essential for high- copy DOA4 to suppress CPY-Inv secretion caused by the loss of Bro1 function. Because high-copy DOA4 partially suppressed the bro1 phenotype, we determined whether the loss of any class E Vps protein could be bypassed by DOA4 overexpression. We transformed either the empty library vector or the library plasmid containing DOA4 into strains in which individual class E VPS genes had been deleted, and then measured the percentage of secreted CPY-Inv (Table I). Suppression failed to occur in strains in which components of the ESCRT-I, -II, or -III complexes had been deleted (Table I). High-copy DOA4 also could not suppress the loss of other class E Vps proteins, including Vps27 (Table I), which functions upstream of the ESCRT-I complex in the recognition of ubiquitinated MVB cargo proteins (Bilodeau et al., 2003; Katzmann et al., 2003). Interestingly, DOA4 overexpression suppressed the secretion of CPY-Inv by vps4 cells but could Figure 3. DOA4 overexpression in bro1 mutant cells suppresses CC CPY-Inv secretion. Schematic diagram of the trafficking of CPY in not suppress either bro1 vps4 or bro1 vps4 double wild-type cells (A) and in bro1 mutant cells (B). Not depicted in A mutant cells (Table I). These observations suggest that high- is the fusion of the endosome/MVB with the vacuole. PM, plasma copy DOA4 suppresses the CPY sorting defects in bro1 and membrane. (C–E) Quantitation of CPY-Inv secretion by BHY10, vps4 mutants by different mechanisms. GOY57, and KGY1 cells transformed either with the empty high- copy (2 ) plasmid (C), the 2  plasmid containing the wild-type C/S High-copy DOA4 suppresses CPS deubiquitination and DOA4 gene (D), or the 2  plasmid containing the mutant doa4 CC allele (E). The bar graph depicts the mean  standard error from sorting defects in bro1 mutant cells multiple independent experiments (Table I). We had hypothesized that a high-copy suppressor of the ab- errant secretion of CPY-Inv by bro1 mutant cells would re- store normal function of the MVB pathway. Indeed, when ibly suppress CPY-Inv secretion. Nucleotide sequence analy- CC sis revealed that this suppressor plasmid contained a 7.3-kb we examined the localization of GFP-CPS in bro1 cells overexpressing DOA4, most of the fusion protein was seen fragment of chromosome IV that includes the complete ORF of five genes, one of which is DOA4. inside the vacuole lumen, with only a small amount detected on the vacuole membrane (Fig. 4 A). Fluorescent punctate The DOA4 gene product is a member of the Ub-specific processing protease (UBP) family of Ub thiolesterases (Wing, structures were not evident in these cells, suggesting that class E compartment formation was significantly reduced. 2003). Doa4 is the UBP required for removal of Ub from the cytoplasmic domain of CPS and other MVB cargo pro- We also observed more GFP-CPS within the vacuole lumen of bro1 cells overexpressing DOA4 (Fig. 4 B) compared teins (Dupre and Haguenauer-Tsapis, 2001; Katzmann et al., 2001; Losko et al., 2001), and subcloning confirmed with bro1 cells alone (Fig. 1 B). However, compared with CC bro1 cells that overexpress DOA4 (Fig. 4 A), much more that DOA4 is the gene within the library plasmid responsible CC for suppression of the bro1 phenotype (unpublished GFP-CPS in bro1 cells overexpressing DOA4 was seen at the vacuole membrane and at structures that resembled class data). The suppression of CPY-Inv secretion mediated by DOA4 overexpression is shown in Fig. 3 D and Table I. High- E compartments (Fig. 4 B). Thus, high-copy DOA4 is much less efficient at suppressing the defect in GFP-CPS sorting in copy DOA4 reduced the percentage of CPY-Inv secreted by CC CC bro1 cells by approximately threefold. In contrast, DOA4 bro1 compared with bro1 cells. Although the overexpression of DOA4 in vps4 cells sup- overexpression resulted in approximately threefold more CPY-Inv secreted by wild-type cells, indicating that high- pressed CPY-Inv secretion (Table I), it did not restore the sorting of GFP-CPS into the vacuole lumen. As shown in copy DOA4 caused a subtle dominant-negative phenotype. Surprisingly, DOA4 overexpression reduced by approxi- Fig. 4 D, GFP-CPS was mislocalized to the vacuole mem- Bro1 recruits Doa4 to endosomes | Luhtala and Odorizzi 721 Table I. Effect of DOA4 over-expression on CPY-Inv secretion and GFP-CPS sorting / 2 µ Percentage of a b Strain DOA4 secreted CPY-Inv GFP-CPS localization c d e 1.0  2.1 (5) VL Wild-type 2.8  1.5 (4) VL 11.5  4.7 (24) VM  EC ∆ CC bro1 3.7  2.5 (7) VL 13.0  3.8 (6) VM  EC bro1∆ 5.6  2.4 (5) VL  VM  EC 39.4  2.5 (3) VM  EC vps4∆ 5.2  1.7 (3) VM  EC 43.7  6.9 (3) VM  EC bro1∆ vps4∆ 46.0  7.1 (3 VM  EC 35.2  2.9 (3) VM  EC ∆ CC bro1 vps4∆ 35.9  4.1 (3) VM  EC ESCRT-I mutants 20.0  5.9 (3) VM  EC vps23∆ 17.6  2.0 (3) VM  EC 13.4  1.2 (3) VM  EC vps28∆ 21.7  4.2 (3) VM  EC 16.3  1.7 (3) VL vps37∆ 15.8  1.3 (3) VL ESCRT-II mutants 20.1  4.5 (3) VM  EC vps22∆ 20.5  6.1 (3) VM  EC 23.1  1.0 (3) VM  EC vps25∆ 20.7  0.9 (3) VM  EC 24.2  6.3 (3) VM  EC vps36∆ 20.6  4.1 (3) VM  EC ESCRT-III mutants 25.2  5.0 (3) VM  EC vps2∆ 27.8  6.5 (3) VM  EC 29.1  4.3 (3) VM  EC vps24∆ 30.9  4.4 (3) VM  EC 24.2  2.9 (3) VM  EC vps20∆ 20.1  6.5 (3) VM  EC 25.8  5.0 (3) VM  EC snf7∆ 24.6  1.1 (3) VM  EC Other class E vps mutants tested 28.0  1.5 (3) VM  EC vps27∆ 26.5  2.6 (3) VM  EC 41.8  13.0 (3) VL nhx1∆ 39.1  13.0 (3) VL vps60∆  17.7  1.3 (3) VM  EC 19.0  5.5 (3) VM  EC Strains were transformed with the empty 2 µ vector () or the 2 µ vector containing DOA4 (). The percentage of total cellular CPY-Inv secreted by cells. Mean  standard error. Number of independent experiments. Vacuole lumen. Vacuole membrane and class E compartment. There was no noticeable defect in GFP-CPS sorting in vps37 and nhx1 cells. brane and the class E compartment in vps4 cells overexpress- Bro1 function. Because the deubiquitination of CPS is nor- ing DOA4, which is identical to the mislocalization of GFP- mally efficient, Ub-CPS was difficult to detect in wild-type CPS in vps4 cells (Odorizzi et al., 1998). High-copy DOA4 cells, but a significant amount of Ub-CPS was observed in CC also failed to restore the sorting of GFP-CPS via the MVB bro1 cells (Fig. 4 E). As shown in Fig. 4 (E and F), overex- pathway in other class E vps mutants (Table I). pression of DOA4 completely restored CPS deubiquitination CC In doa4 mutant cells, CPS and other MVB cargoes accu- in bro1 cells, which is consistent with a role for Doa4 in mulate in their ubiquitinated forms (Dupre and Haguenauer- deubiquitinating MVB cargo proteins. Tsapis, 2001; Katzmann et al., 2001; Losko et al., 2001). We Interestingly, much more Ub-CPS was observed in bro1 CC recently showed that ubiquitinated CPS (Ub-CPS) also accu- cells compared with the level of Ub-CPS seen in bro1 mulates in bro1 cells (Odorizzi et al., 2003). Therefore, we cells (Fig. 4 E), indicating that the deubiquitination of CPS investigated whether DOA4 overexpression could alleviate is more severely compromised if the Bro1 protein is absent. the defect in CPS deubiquitination that occurs upon loss of Furthermore, the amount of Ub-CPS in bro1 mutants 722 The Journal of Cell Biology | Volume 166, Number 5, 2004 Figure 5. DOA4 overexpression restores MVB morphology in bro1 CC mutant cells. (A) EM showing MVBs in BHY10 cells. Arrowheads Figure 4. DOA4 overexpression in bro1 mutant cells restores indicate MVBs. n, nucleus. Bar, 0.2 m. (B) MVBs from the boxed the sorting and deubiquitination of CPS. (A–D) Fluorescence and area shown in A. Bar, 0.1 m. (C) EM showing a typical class E DIC microscopy of GOY57 (A), KGY1 (B), BHY10 (C), and DBY42 compartment in GOY57 cells. Bar, 0.1 m. (D) EM showing multi- (D) cells expressing the GFP-CPS fusion and transformed with 2 vesicular structures in GOY57 cells transformed with 2  DOA4. DOA4. Arrowheads indicate class compartments. Bars, 2.5 m. The arrowhead indicates a tubule projecting from an MVB toward (E) Immunoprecipitates of CPS were examined by Western blotting the cytoplasm; note that the membrane bilayer of the tubule is using anti-Ub or anti-CPS antibodies. Note that the lumenal domain continuous with the bilayer surrounding the lumenal vesicles. Bar, of CPS is differentially glycosylated and is observed as a doublet. 0.1 m. (E) MVBs and class E compartments were counted in 200 (F) Quantitation of the intensities of Ub-CPS versus CPS present cells which had a diameter  2.5 m. Each value is indicated above in each lane in E. Bar graph depicts mean values  SD from two its respective bar in the graph and was expressed as the number per independent experiments. 200 cells. The multivesicular structures that had tubular projections (D, arrowhead) were counted as MVBs. was much less significantly reduced upon overexpression of DOA4, in contrast with the complete restoration of CPS CC High-copy DOA4 restores MVB morphology in deubiquitination that occurred in bro1 cells. This result bro1 mutant cells is consistent with our observation that high-copy DOA4 was much more effective at suppressing the GFP-CPS sort- By fluorescence microscopy, it appeared that the overexpres- CC ing defect in bro1 cells (Fig. 4 A) versus bro1 cells (Fig. sion of DOA4 caused a reduction in the number of class E 4 B). Thus, excess amounts of Doa4 effectively alleviate the compartments in bro1 mutant cells. Therefore, we used EM mutant phenotype caused by deletion of the Bro1 coiled- to examine the effect that high-copy DOA4 has on endoso- coil domain but cannot substitute for deletion of the entire mal ultrastructure. In wild-type cells, we readily observed Bro1 protein. MVBs, with each compartment consisting of a limiting Bro1 recruits Doa4 to endosomes | Luhtala and Odorizzi 723 tures, examples of which are shown in Fig. 5 D. These com- partments consisted of a limiting membrane bilayer that en- circled multiple vesicular profiles within the compartment lumen, which is similar to MVBs in wild-type cells (Fig. 5 B). Quantitative analysis indicated that DOA4 overexpres- CC sion in bro1 cells caused a dramatic reduction in the number of class E compartments and resulted in almost as many MVBs as we had observed in wild-type cells (Fig. 5 E). The apparent reformation of MVBs and the concomitant disappearance of class E compartments upon DOA4 overex- pression occurred to a lesser extent in bro1 cells (Fig. 5 E), again indicating that high-copy DOA4 is not as efficient at suppressing the phenotype caused by a total loss of Bro1 compared with its ability to suppress the phenotype caused by a deletion of only the Bro1 coiled-coil domain. Interestingly, the overexpression of DOA4 in bro1 mutant cells occasionally resulted in multivesicular compartments having tubules projecting toward the cytoplasm that resemble the cisterna-like structures of class E compartments (Fig. 5 D, arrowhead). Although further studies are needed in order to establish the identity of these unusual structures, they may correspond to intermediate MVB/class E compartments. The localization of Doa4 to endosomes requires Bro1 Doa4 had previously been shown to associate with endoso- mal compartments (Amerik et al., 2000). Indeed, when we integrated a DOA4-GFP gene fusion in place of the wild- type DOA4 gene in cells that have normal Vps4 function (VPS4 ), we observed multiple fluorescent punctate struc- tures in addition to diffuse cytoplasmic fluorescence (Fig. 6 A). This pattern of fluorescence resembles the intracellular localization of Bro1-GFP in VPS4 cells (Fig. 2 A). Also like Bro1-GFP, Doa4-GFP was concentrated at class E compart- ments in vps4 cells (Fig. 6 B). Doa4-GFP was also localized CC CC to class E compartments in bro1 and bro1 vps4 cells (Fig. 6, C and D, closed arrowheads), indicating that the Bro1 coiled-coil domain is not required for the association of Doa4 with late endosomes. Interestingly, Doa4-GFP was localized to additional punctate structures that were not la- beled by FM 4-64 in VPS4 and vps4 cells expressing the CC mutant bro1 allele in place of the wild-type BRO1 gene (Fig. 6, C and D, open arrowheads), which is similar to the CC localization pattern of the mutant bro1 -GFP protein (Fig. Figure 6. Bro1 is required for the localization of Doa4 to endosomes. 2, C and D). Fluorescence and DIC microscopy of GOY74 (A), GOY75 (B), GOY88 (C), GOY93 (D), GOY82 (E), and GOY83 (F) cells stained A dramatic change in the localization of Doa4-GFP oc- with FM 4-64. Closed arrowheads indicate colocalization of GFP curred in cells in which the BRO1 gene had been deleted. and FM 4-64 at class E compartments. In C and D, open arrowheads Both in bro1 cells (Fig. 6 E) and bro1 vps4 double mu- indicate GFP-positive structures not labeled by FM 4-64. In E and F, tant cells (Fig. 6 F), Doa4-GFP failed to localize to class E open arrowheads indicate FM 4-64–positive class E compartments compartments and was, instead, diffusely distributed. Some at which Doa4-GFP is not localized. Bars, 2.5 m. faintly fluorescent structures were evident at the periphery of these cells but are unlikely to be endosomes, as they were membrane bilayer surrounding numerous vesicular profiles never stained when FM 4-64 was incubated continuously (Fig. 5, A and B). In contrast, MVBs were never evident in with cells (unpublished data), a procedure that results in CC bro1 or bro1 cells, which, instead, contained numerous staining of all compartments of the endocytic pathway (Vida class E compartments. An example of a class E compartment and Emr, 1995). Western blot analysis confirmed that the CC seen in bro1 cells is shown in Fig. 5 C. Similar structures expression of full-length Doa4-GFP in bro1 and bro1 were also seen in bro1 cells (not depicted). vps4 double mutant cells was equivalent to its expression High-copy DOA4 caused a striking change in endosomal in wild-type and vps4 cells (Fig. S1, available at http:// CC morphology in bro1 mutant cells. Rather than class E www.jcb.org/cgi/content/full/jcb.200403139/DC1), indicat- compartments, we observed numerous multivesicular struc- ing that the loss of punctate localization of Doa4-GFP in the 724 The Journal of Cell Biology | Volume 166, Number 5, 2004 Figure 7. Doa4 is not required for the localization of Bro1 to endosomes. Fluorescence and DIC microscopy of GOY69 (A) and GOY70 (B) cells stained with FM 4-64. Arrowheads indicate class E compartments. Bars, 2.5 m. absence of Bro1 was not due to aberrant cleavage of the fu- sion protein. Furthermore, transformation of a plasmid en- CC Figure 8. Doa4 interacts with Bro1 and the mutant Bro1 protein coding the wild-type BRO1 gene into bro1 and bro1 on membranes. (A) Detergent extracts of total lysates from GOY107, vps4 cells restored the localization of Doa4-GFP to endo- GOY108, GOY106, and GOY105 cells were subjected to immuno- somal compartments (unpublished data). Thus, Bro1 is es- precipitation with rabbit anti-GFP polyclonal antiserum under native sential for the localization of Doa4 to endosomes. conditions and examined by Western blotting. (B) Cell lysates were separated into membrane pellet (P) and soluble (S) fractions and To test whether the endosomal localization of Bro1 is like- extracted with detergent before immunoprecipitation and Western wise dependent on Doa4, we examined the localization of blot analysis. Bro1-GFP in cells in which the DOA4 gene had been de- leted. In doa4 VPS4 cells, we observed punctate fluores- CC Bro1-HA and bro1 -HA coimmunoprecipitated with Doa4- cent structures in addition to diffuse cytoplasmic fluores- GFP from the membrane fraction of VPS4 cells; however, cence (Fig. 7 A), whereas in doa4 vps4 double mutant these interactions were more significantly enriched in mem- cells, the GFP fluorescence was concentrated at class E com- brane extracts considering that the vast majority of Doa4- partments (Fig. 7 B). This pattern of fluorescence was virtu- GFP in VPS4 cells was recovered from the soluble fraction ally identical to the intracellular localization of Bro1-GFP in (Fig. 8 B). In vps4 cells, the interaction between Doa4- VPS4 and vps4 cells (Fig. 2). Therefore, Doa4 is not re- CC GFP and both Bro1-HA and bro1 -HA occurred almost quired for the endosomal localization of Bro1. exclusively in the membrane fraction, again despite the fact that more Doa4-GFP was immunoprecipitated from the sol- Doa4 interacts with Bro1 uble fraction (Fig. 8 B). Altogether, these results indicate Because Bro1 is required for the endosomal localization of that Doa4 associates with Bro1 on membranes and that the Doa4, we investigated whether Doa4 physically interacts coiled-coil domain is not required for this interaction to with Bro1 by immunoprecipitating the Doa4-GFP fusion occur. protein from detergent-solubilized extracts of yeast cell lysates under native conditions. As shown in Fig. 8 A, CC Endosomal localization of Doa4 in ESCRT-III Bro1-HA and bro1 -HA fusion proteins coimmunopre- mutant cells cipitated with Doa4-GFP from total extracts of VPS4 and vps4 cells. Control immunoprecipitations from lysates of A previous study had suggested that the association of Doa4 strains not expressing DOA4-GFP confirmed that the isola- with endosomes was dependent on two other class E Vps pro- tion of Bro1-HA using anti-GFP antiserum required Doa4 teins, Vps24 and Snf7 (Amerik et al., 2000). Both Vps24 and (Fig. S2, available at http://www.jcb.org/cgi/content/full/ Snf7 are components of the ESCRT-III complex that oligo- jcb.200403139/DC1). Furthermore, the interaction between merizes on endosomal membranes (Babst et al., 2002a). We Bro1-HA and Doa4-GFP also occurred in anti-HA immu- recently showed that the endosomal localization of Bro1 re- noprecipitations followed by anti-GFP Western blots (un- quires Snf7 but not Vps24, suggesting that Snf7 has a role in published data). the recruitment or stabilization of Bro1 at endosomes (Odo- We tested whether the interaction between Doa4 and rizzi et al., 2003). Therefore, we examined the localization of Bro1 occurred on membranes and/or in the cytoplasm by Doa4-GFP in vps4 cells in which either VPS24 or SNF7 centrifuging cell lysates before immunoprecipitation in or- had been deleted. In contrast with the previous analysis of der to generate a pellet fraction enriched in late endosomal Doa4 localization (Amerik et al., 2000), we observed Doa4- membranes and a supernatant fraction containing soluble GFP at class E compartments in vps24 vps4 double mu- proteins (Odorizzi et al., 2003). As shown in Fig. 8 B, more tant cells (Fig. 9 A). However, Doa4-GFP was diffusely dis- Bro1 recruits Doa4 to endosomes | Luhtala and Odorizzi 725 Figure 10. Model for the recruitment of Doa4 to endosomes by Bro1. Bro1 is required for the association of Doa4 with endosomes, where Doa4 catalyzes deubiquitination of MVB cargo proteins. The endosomal recruitment of Bro1, and consequently the recruit- ment of Doa4, is dependent on Snf7, which oligomerizes on endosomal membranes with the other ESCRT-III components (Vps2, Vps24, and Vps20). After their deubiquitination, MVB cargoes are sorted into lumenal vesicles. Not depicted is the dissociation of Bro1, Doa4, and ESCRT-III proteins from endosomes, which is catalyzed by the Vps4 ATPase. Recruitment of Doa4 by Bro1 Both Doa4-GFP and Bro1-GFP were associated with endoso- mal compartments, but upon deletion of the BRO1 gene, the localization of Doa4-GFP was shifted to the cytoplasm. In contrast, Bro1-GFP remained associated with endosomes re- gardless of whether the DOA4 gene had been deleted. Native Figure 9. Localization of Doa4-GFP in ESCRT-III mutants. (A and B) immunoprecipitation experiments revealed a physical interac- Fluorescence and DIC microscopy of GOY113 (A) and GOY114 (B) tion between Doa4 and Bro1, which could reflect direct bind- cells stained with FM 4-64. Closed arrowheads indicate colocalization ing of Doa4 by Bro1 or, alternatively, could be mediated by of Doa4-GFP and FM 4-64 at class E compartments. Open arrow- heads indicate class E compartments at which Doa4-GFP is not another component that binds both proteins. In either case, localized. Bars, 2.5 m. (C) Native immunoprecipitations from the association of Doa4 with Bro1 occurred predominantly in detergent extracts of total lysates of GOY115 and GOY116 cells. subcellular fractions containing late endosomal membranes, which is consistent with the ordered recruitment of Bro1 to endosomes followed by the recruitment of Doa4 (Fig. 10). tributed in snf7 vps4 double mutants (Fig. 9 B). This pat- tern in the localization of Doa4-GFP was identical to the Consistent with this model, the association of Doa4-GFP with endosomes required Snf7, a component of the ESCRT- localization of Bro1 that we had previously observed in these same mutant strains (Odorizzi et al., 2003). The inability of III complex. We recently showed that Snf7 is essential for the endosomal localization of Bro1 (Odorizzi et al., 2003), and Doa4-GFP to localize to endosomal compartments in the ab- sence of Snf7 may, therefore, be due to the requirement for in mammalian cells, the orthologue of Bro1, Alix/Aip1, binds Chmp4b, which is an orthologue of Snf7 (Katoh et al., Snf7 in the endosomal localization of Bro1 (Odorizzi et al., 2003). Indeed, as shown in Fig. 9 C, neither Vps24 nor Snf7 2003; Martin-Serrano et al., 2003; Strack et al., 2003; von Schwedler et al., 2003; Peck et al., 2004). Thus, it is likely coimmunoprecipitated with Doa4 from VPS4 or vps4 cell extracts. Thus, the ESCRT-III complex does not appear to that Snf7 has an indirect role in the endosomal localization of Doa4 by functioning to recruit Bro1 to endosomes or to sta- have a direct role in the recruitment of Doa4 to endosomes. bilize the association of Bro1 with endosomal membranes. In- deed, we were unable to detect a physical interaction between Discussion Doa4 and either Snf7 or Vps24, another component of the Protein sorting in the MVB pathway requires Bro1, a cyto- ESCRT-III complex. Furthermore, high-copy DOA4 could plasmic protein that associates with endosomal compart- not suppress the defects in either CPY or CPS sorting caused ments (Odorizzi et al., 2003). Here, we show that DOA4 by the loss of any ESCRT-III component. overexpression suppresses the defects in MVB transport and It is unlikely that Snf7 recruits Bro1 to endosomes by in- restores the multivesicular morphology of endosomes in teracting with the Bro1 coiled-coil domain, as this region of bro1 mutant cells. These observations can be explained by Bro1 was not required for its association with endosomal our finding that Bro1 associates with Doa4 on endosomal membranes. Furthermore, despite the fact that we identified CC membranes and that the recruitment of Doa4 to endosomes DOA4 in a screen for high-copy suppressors of the bro1 requires Bro1. Thus, our results indicate a key role for Bro1 mutant phenotype, the Bro1 coiled-coil domain is not nec- in regulating the timing and location of Doa4 activity in the essary for Bro1 to interact with Doa4. Nevertheless, the MVB pathway. coiled-coil domain is important for Bro1 function, as the 726 The Journal of Cell Biology | Volume 166, Number 5, 2004 CC transport of GFP-CPS was blocked in bro1 mutant cells Consistent with a role for Bro1 in coordinating Doa4 as effectively as it was in bro1 cells. Therefore, the recruit- function are the previous observations that Bro1 is re- ment of Doa4 to endosomes is only one aspect of the func- quired for the deubiquitination of CPS (Odorizzi et al., tion of Bro1 in the MVB pathway. The role of the Bro1 2003) as well as Gap1, an amino acid permease at the coiled-coil domain is not yet clear. Thus far, we have been plasma membrane that is down-regulated by endocytosis unable to identify another protein that interacts with this re- and sorted via the MVB pathway (Nikko et al., 2003). In- gion of Bro1, and we have been unable to detect homo-oli- terestingly, in bro1 mutant cells, Gap1 is recycled to the gomerization of Bro1 that could be mediated by the coiled- plasma membrane but, unlike CPS, does not accumulate coil domain (unpublished data). in its ubiquitinated form. However, ubiquitinated Gap1 is readily detected in bro1 mutant cells if recycling is blocked, Suppression by high-copy DOA4 suggesting that in the absence of Bro1, the deubiquitina- The restoration of multivesicular structures upon the overex- tion of Gap1 (and possibly other endocytic cargoes) occurs pression of DOA4 coincided with the disappearance of class elsewhere within the cell and may be catalyzed by one of E compartments, which presumably enables the CPY recep- the 15 other UBPs in yeast (Nikko et al., 2003). tor, Vps10, to recycle to the Golgi where it binds newly syn- The suppressor activity of high-copy DOA4 was abol- thesized CPY-Inv in order to transport this soluble cargo ished if the putative active-site cysteine residue was altered protein to the endosome (Cereghino et al., 1995; Cooper to serine, indicating that suppression most likely occurred and Stevens, 1996). Accordingly, we have observed that a through substrate deubiquitination. However, the deubiq- Vps10-GFP fusion protein was not concentrated at class E uitination of cargoes is not essential for MVB sorting be- compartments in bro1 mutant cells overexpressing DOA4 cause chimeric cargo proteins that are expressed as transla- but was, instead, localized to multiple punctate structures tional fusions to Ub cannot be deubiquitinated yet are (unpublished data), which is similar to its localization in transported efficiently via the MVB pathway (Reggiori and wild-type cells (Burda et al., 2002; Odorizzi et al., 2003). Pelham, 2001; Urbanowski and Piper, 2001; Bilodeau et Interestingly, although high-copy DOA4 restored the mul- al., 2003). Nevertheless, Doa4 is required for the MVB tivesicular morphology of late endosomes and suppressed pathway, as the sorting of MVB cargo proteins is blocked CC CPY-Inv secretion by both bro1 and bro1 cells, the in doa4 mutant cells (Losko et al., 2001; Reggiori and Pel- transport of GFP-CPS via the MVB pathway was efficiently ham, 2001). In addition to MVB cargoes, Doa4 could CC restored by DOA4 overexpression only in bro1 and not deubiquitinate a component of the MVB sorting machin- bro1 cells. Furthermore, the overexpression of DOA4 in ery in order to regulate its activity. It is not known whether bro1 cells did not significantly enhance the deubiquitina- class E Vps proteins in yeast are regulated by ubiquitina- tion of CPS, whereas the minor amount of Ub-CPS seen in tion. In mammalian cells, however, several cytoplasmic CC the bro1 strain was completely eliminated by DOA4 proteins that control receptor down-regulation are mono- overexpression. These observations are consistent with the ubiquitinated, including Hrs, which is the orthologue of ability of Doa4 to be recruited to endosomes in cells express- the yeast class E Vps protein, Vps27 (Polo et al., 2002). CC ing the mutant bro1 protein but not in cells in which CIN85 and endophilin are also monoubiquitinated cy- Bro1 is completely absent. Moreover, these findings suggest toplasmic components in mammalian cells that associate that restoring MVB morphology alone by DOA4 overex- with endosomes (Haglund et al., 2002; Angers et al., pression is not sufficient for a complete restoration of the 2004), and both proteins interact with Alix/Aip1 (Vito et MVB pathway. The overexpression of DOA4 in bro1 cells al., 1996; Chatellard-Causse et al., 2002). Like Bro1 in may result in excess amounts of Doa4 that could deubiquiti- yeast, Alix/Aip1 could recruit a UBP that deubiquitinates nate proteins which regulate the formation of MVB vesicles, CIN85 and endophilin in order to regulate their function but the sorting of cargoes into MVB vesicles per se may be and/or localization. critically dependent on the coordination of Doa4 on endo- Our results suggest a model in which Bro1 recruits Doa4 somal membranes by Bro1 as well as other aspects of Bro1 to endosomal membranes, thereby controlling the timing function in the MVB pathway. and location of Doa4 activity in the MVB pathway (Fig. 10). The association of Bro1 itself with endosomes occurs Deubiquitination in the MVB pathway after the assembly of the ESCRT-III complex on endo- Ubiquitination is tightly controlled by Ub-conjugation en- somes (Odorizzi et al., 2003). Similarly, the deubiquitina- zymes and is counterbalanced by deubiquitination. Both tion of MVB cargo proteins occurs downstream of the poly- and monoubiquitinated proteins undergo deubiqui- functions of the ESCRT-I, -II, and -III complexes (Nikko tination, but few Ub thiolesterases have been assigned to et al., 2003) but before enclosure of cargoes within MVB specific substrates (Wing, 2003). There are 16 UBPs in S. vesicles (Hicke and Dunn, 2003). Thus, the Bro1-depen- cerevisiae, suggesting that many of these enzymes have dent coordination of Doa4 on endosomal membranes and highly specific and regulated functions. However, Doa4 the subsequent deubiquitination of cargo proteins (and probably has a wide variety of substrates. In addition to its possibly MVB sorting components) is likely to be one of role in the removal of monoubiquitin from MVB cargo the last steps in the MVB pathway, ensuring that cargo proteins, Doa4 is likely to function in the removal of poly- proteins are concentrated at regions of the endosomal ubiquitin from proteasomal substrates, as Doa4 copurifies membrane where invagination occurs. In mammalian cells, with proteasomes that have been isolated from yeast cell the assembly/budding of HIV-1 depends on ubiquitina- extracts (Papa et al., 1999). tion of the viral Gag protein (for review see Pornillos et al., Bro1 recruits Doa4 to endosomes | Luhtala and Odorizzi 727 CC allele, the SpeI–SalI fragment containing the To construct the bro1 2002), but a role for deubiquitination in this process has BRO1 gene in plasmid pGO187 (Odorizzi et al., 2003) was subcloned not been described. However, recent studies indicate that into SpeI–SalI-digested pRS413, resulting in plasmid pGO263, which Alix/Aip1 interacts with both the ESCRT-I and -III com- was digested with EcoRV and recircularized using T4 DNA ligase. The CC allele consist- plexes and is involved in viral escape from host cells (Strack resulting plasmid (pGO265) contained the mutant bro1 ing of codon 540 adjoined to codon 586. The SpeI–SalI fragment in et al., 2003; von Schwedler et al., 2003). Future studies pGO265 was subcloned into SpeI–SalI-digested pRS416, pRS426, and may determine whether Alix/Aip1 has a role analogous to pRS306, resulting in pGO273, pGO272, and pGO287, respectively. The CC Bro1 and functions in the recruitment of a specific UBP to allele was integrated into the genome in place of the wild-type bro1 BRO1 gene using pGO287 by homologous recombination (Guthrie and the site of viral budding. C/S allele was constructed by PCR and subcloned Fink, 2002). The doa4 into pRS202, resulting in pGO309. Materials and methods Microscopy Yeast strains and plasmid constructions GFP and FM 4-64 fluorescence and differential interference contrast (DIC) Standard protocols were used for culturing S. cerevisiae, cellular transfor- microscopy was performed using a DMRXA microscope (Leica) equipped mations, and spheroplast preparations (Guthrie and Fink, 2002). See Table with a Cooke Sensicam digital camera (Applied Scientific Instruments). Im- II for the genotypes of yeast strains used in this work. Gene deletions were ages were deconvolved using Slidebook software (Intelligent Imaging In- constructed by homologous recombination using site-specific deletion cas- novations) and processed using Adobe Photoshop 7.0 software (Adobe settes (Guthrie and Fink, 2002). Systems Inc.). Cells were stained with FM 4-64 using a pulse-chase proce- Table II. Yeast strains used in this work Strain Genotype Reference SEY6210 MAT leu2-3,112 ura3-52 his3∆ 200 trp1-∆ 901 lys2-∆ 801 suc2-∆ 9 Robinson et al., 1988 BHY10 SEY6210; leu2-3,112::pBHY11 Horazdovsky et al., 1994 ∆ CC GOY57 BHY10; bro1 This work KGY1 BHY10; bro1∆ ::HIS3 This work DMY1 SEY6210; BRO1-GFP::HIS3 This work DMY2 SEY6210; BRO1-GFP::HIS3 vps4∆ ::TRP1 This work ∆ CC DBY15 BHY10; bro1 -GFP::HIS3 This work ∆ CC DBY18 BHY10; bro1 -GFP::HIS3 vps4∆ ::TRP1 This work GOY23 SEY6210; pep4∆ ::LEU2 prb1∆ ::LEU2 This work ∆ CC GOY71 GOY23; bro1 This work DBY43 GOY23; bro1∆ ::HIS3 This work GOY74 SEY6210; DOA4-GFP::HIS3 This work GOY75 SEY6210; DOA4-GFP::HIS3 vps4∆ ::TRP1 This work ∆ CC GOY88 SEY6210; DOA4-GFP::HIS3 bro1 This work ∆ CC GOY93 SEY6210; DOA4-GFP::HIS3 bro1 vps4∆ ::TRP1 This work GOY82 SEY6210; DOA4-GFP::HIS3 bro1∆ ::KAN This work GOY83 SEY6210; DOA4-GFP::HIS3 bro1∆ ::KAN vps4∆ ::TRP1 This work GOY69 SEY6210; BRO1-GFP::HIS3 doa4∆ ::KAN This work GOY70 SEY6210; BRO1-GFP::HIS3 doa4∆ ::KAN vps4∆ ::TRP1 This work GOY113 SEY6210; DOA4-GFP::KAN vps4∆ ::TRP1 vps24∆ ::HIS3 This work GOY114 SEY6210; DOA4-GFP::KAN vps4∆ ::TRP1 snf7∆ ::HIS3 This work GOY107 GOY23; DOA4-GFP::KAN BRO1-HA::HIS3 This work GOY108 GOY23; DOA4-GFP::KAN BRO1-HA::HIS3 vps4∆ ::TRP1 This work ∆ CC GOY106 GOY23; DOA4-GFP::KAN bro1 -HA::HIS3 This work ∆ CC GOY105 GOY23; DOA4-GFP::KAN bro1 -HA::HIS3 vps4∆ ::TRP1 This work DBY42 BHY10; vps4∆ ::TRP1 This work NLY25 BHY10; bro1∆ ::HIS3 vps4∆ ::TRP1 This work ∆ CC DBY24 BHY10; bro1 vps4∆ ::TRP1 This work GOY86 BHY10; vps23∆ ::HIS3 This work NLY10 BHY10; vps28∆ ::HIS3 This work NLY8 BHY10; vps37∆ ::HIS3 This work GOY85 BHY10; vps22∆ ::HIS3 This work GOY78 BHY10; vps25∆ ::HIS3 This work CRY2 BHY10; vps36∆ ::HIS3 This work GOY79 BHY10; vps2∆ ::HIS3 This work CRY1 BHY10; vps24∆ ::HIS3 This work GOY80 BHY10; vps20∆ ::HIS3 This work GOY81 BHY10; snf7∆ ::HIS3 This work GOY87 BHY10; vps27∆ ::HIS3 This work GOY91 BHY10; nhx1∆ ::HIS3 This work GOY90 BHY10; vps60∆ ::HIS3 This work 728 The Journal of Cell Biology | Volume 166, Number 5, 2004 dure at 30C as described previously (Odorizzi et al., 2003). 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The ALG-2-interacting protein Alix associates Online supplemental material with CHMP4b, a human homologue of yeast Snf7 that is involved in multi- Fig. S1 shows that there is not a significant difference in the expression vesicular body sorting. J. Biol. Chem. 278:39104–39113. levels of full-length Doa4-GFP in the strains shown in Fig. 6. Fig. S2 Katzmann, D.J., M. Babst, and S.D. Emr. 2001. Ubiquitin-dependent sorting into shows the results from a negative control for Fig. 8 in which Bro1-HA is the multivesicular body pathway requires the function of a conserved endo- not immunoprecipitated under native conditions by anti-GFP antiserum somal protein sorting complex, ESCRT-I. Cell. 106:145–155. in the absence of Doa4-GFP expression. Online supplemental material is Katzmann, D.J., C.J. Stefan, M. Babst, and S.D. Emr. 2003. Vps27 recruits ESCRT available at http://www.jcb.org/cgi/content/full/jcb.200403139/DC1. machinery to endosomes during MVB sorting. J. Cell Biol. 162:413–423. Losko, S., F. Kopp, A. Kranz, and R. Kolling. 2001. Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 We thank Jeff Kimes and Tom Giddings for EM, Monica Darland for con- mutant. Mol. Biol. Cell. 12:1047–1059. struction of plasmids, Doug Burch, David Mellman, Caleb Richter, and Martin-Serrano, J., A. Yarovoy, D. Perez-Caballero, and P.D. Bieniasz. 2003. Di- Kelly Geiger for construction of yeast strains (University of Colorado), and vergent retroviral late-budding domains recruit vacuolar protein sorting fac- Charles Zuker for providing rabbit anti-GFP polyclonal antiserum. tors by using alternative adaptor proteins. Proc. Natl. Acad. Sci. USA. 100: G. Odorizzi is supported by an American Cancer Society grant (RSG- 12414–12419. 02-147-01) and a National Institutes of Health grant (GM065505). Matsuo, H., J. Chevallier, N. Mayran, I. Le Blanc, C. Ferguson, J. Faure, N.S. Blanc, S. Matile, J. Dubochet, R. Sadoul, et al. 2004. 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Sorting of proteins into multivesicular bodies: along the ubiquitin-proteasome pathway. Int. J. Bioch. Cell Biol. 35:590–605. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Pubmed Central

Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes

Luhtala, Natalie; Odorizzi, Greg
The Journal of Cell Biology , Volume 166 (5) – Aug 30, 2004
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Copyright © 2004, The Rockefeller University Press
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Abstract

JCB Article Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes Natalie Luhtala and Greg Odorizzi Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 biquitination directs the sorting of cell surface recep- of DOA4 restores cargo protein deubiquitination and sorting via the MVB pathway and reverses the abnormal endo- tors and other integral membrane proteins into the multivesicular body (MVB) pathway. Cargo proteins somal morphology typical of bro1 mutant cells, resulting in the restoration of multivesicular endosomes. We further are subsequently deubiquitinated before their enclosure within MVB vesicles. In Saccharomyces cerevisiae, Bro1 demonstrate that Doa4 interacts with Bro1 on endosomal membranes and that the recruitment of Doa4 to endosomes functions at a late step of MVB sorting and is required for cargo protein deubiquitination. We show that the loss of requires Bro1. Thus, our results point to a key role for Bro1 in coordinating the timing and location of deubiquitination Bro1 function is suppressed by the overexpression of DOA4, which encodes the ubiquitin thiolesterase required for the by Doa4 in the MVB pathway. removal of ubiquitin from MVB cargoes. Overexpression Introduction Multivesicular bodies (MVBs) are late endosomes contain- the linkage of a single Ub subunit (or a chain of two to three ing lumenal vesicles formed by invagination of the limiting subunits) to their cytoplasmic domains (Hicke and Dunn, endosomal membrane. The MVB vesicles are delivered into 2003). Ub is removed from both polyubiquitinated and the hydrolytic lumen of the lysosome/vacuole upon fusion monoubiquitinated proteins by deubiquitinating enzymes, of the limiting MVB membrane with the lysosomal/vacuolar thereby enabling cells to maintain a constant pool of Ub membrane. A variety of cell surface receptors down-regulated (Weissman, 2001; Wing, 2003). from the plasma membrane are sorted into MVB vesicles en The sorting of ubiquitinated MVB cargoes is controlled route to being degraded, including many growth factor re- by class E Vps proteins, a set of conserved cytoplasmic pro- ceptor tyrosine kinases in animals and G protein-coupled teins that associate with endosomal membranes. Several class pheromone receptors in the budding yeast Saccharomyces E Vps proteins coassemble into distinct complexes that bind cerevisiae. In yeast, several biosynthetic enzymes are also sorted to ubiquitinated cargo proteins and guide them into the into MVB vesicles during their transport from the Golgi to MVB pathway. In yeast, cargoes initially bind the Vps27– the vacuole (for review see Hicke and Dunn, 2003). Hse1 complex and subsequently interact with the ESCRT-I Ubiquitination mediates the sorting of integral membrane complex (Vps23, Vps28, and Vps37; Bilodeau et al., 2003; proteins into the MVB pathway. Ubiquitin (Ub) is a highly Katzmann et al., 2003). The ESCRT-II (Vps22, Vps25, and conserved 76-aa polypeptide that is covalently linked to specific Vps36) and ESCRT-III (Vps2, Vps20, Vps24, and Snf7) protein substrates by a cascade of Ub-conjugation enzymes. complexes function downstream of ESCRT-I (Babst et al., Ubiquitination was first characterized to occur on soluble 2002a, b), but their precise roles are not known. In mamma- proteins that are polyubiquitinated via the attachment of a lian cells, class E Vps orthologues also mediate the sorting of chain of four or more Ub subunits, which targets these sub- MVB cargo proteins (Hicke and Dunn, 2003). Further- strates for degradation by the proteasome (Weissman, 2001). more, this machinery is exploited by certain enveloped viruses In contrast, MVB cargo proteins are monoubiquitinated via in order to escape from host cells. For example, Tsg101, the mammalian orthologue of yeast Vps23, is recruited to HIV-1 budding sites at the plasma membrane by directly interacting The online version of this article contains supplemental material. Address correspondence to Greg Odorizzi, Molecular, Cellular, and Developmental Biology, 347 UCB, University of Colorado, Boulder, Abbreviations used in this paper: CPS, carboxypeptidase S; CPY, carboxy- CO 80309-0347. Tel.: (303) 735-0179. Fax: (303) 492-7744. email: peptidase Y; CPY-Inv, CPY-invertase; DIC, differential interference con- [email protected] trast; MVB, multivesicular body; Ub, ubiquitin; UBP, ubiquitin-specific Key words: endosomes; protein sorting; ubiquitin thiolesterase processing protease. © The Rockefeller University Press, 0021-9525/2004/08/717/13 $8.00 The Journal of Cell Biology, Volume 166, Number 5, August 30, 2004 717–729 http://www.jcb.org/cgi/doi/10.1083/jcb.200403139 717 The Journal of Cell Biology 718 The Journal of Cell Biology | Volume 166, Number 5, 2004 with ubiquitinated viral Gag proteins (for review see Pornillos et al., 2002). Additional class E Vps proteins function in the MVB pathway in yeast, including Vps4, an AAA-type ATPase that catalyzes the dissociation of ESCRT complexes from endo- somal membranes (Babst et al., 2002a,b). Vps4 also func- tions in the endosomal dissociation of Bro1, a class E Vps protein required for the deubiquitination of cargo proteins at a late stage of the MVB pathway downstream of the ES- CRT-III complex (Nikko et al., 2003; Odorizzi et al., 2003). The association of Bro1 with endosomes requires Snf7 (Odorizzi et al., 2003), and the mammalian Bro1 ortho- logue, Alix (also known as Aip1), has been shown to bind the mammalian Snf7 orthologue, CHMP4b (Katoh et al., 2003; Martin-Serrano et al., 2003; Peck et al., 2004; Strack et al., 2003; von Schwedler et al., 2003). Studies of HIV-1– infected cells have revealed that Alix/Aip1 also binds Tsg101 during viral budding from the plasma membrane (Strack et al., 2003; von Schwedler et al., 2003), suggesting that Alix/ Figure 1. The coiled-coil domain of Bro1 is essential for CPS transport via the MVB pathway. (A) Schematic diagram of Bro1 Aip1 bridges an interaction between the ESCRT-I and -III indicating the locations of the Bro1 domain (BOD), the coiled-coil complexes. Interestingly, Alix/Aip1 binds specifically to syn- domain (CC), and the proline-rich domain (PRD). (B and C) Fluores- thetic liposomes containing lysobisphosphatidic acid and ap- cence and DIC microscopy of BHY10, GOY57 and KGY1 cells pears to antagonize the ability of this unconventional phos- expressing a GFP-CPS fusion. In C, cells were transformed with a CC pholipid to promote membrane invagination (Matsuo et al., high-copy (2 ) plasmid encoding bro1 or wild-type BRO1. Arrowheads indicate class E compartments (B) and class E com- 2004), but the role of Alix/Aip1 in controlling membrane partment-like structures (C). Bars, 2.5 m. dynamics is not clear. Here, we report that the defects in MVB sorting caused by the loss of Bro1 function in yeast are suppressed by over- expression of the DOA4 gene, which encodes the Ub thio- nome in place of the wild-type BRO1 gene. To determine CC lesterase required for the removal of Ub from MVB cargo whether MVB sorting was functional in bro1 cells, we ex- proteins (Dupre and Haguenauer-Tsapis, 2001; Katzmann amined the intracellular localization of a fusion protein in et al., 2001; Losko et al., 2001). Specifically, our data dem- which GFP was attached to the cytoplasmic domain of car- onstrate that high-copy expression of DOA4 restores cargo boxypeptidase S (CPS), a biosynthetic enzyme that is sorted protein deubiquitination and subsequent transport via the via the MVB pathway during its transport from the Golgi to MVB pathway in bro1 mutant cells. The catalytic activity the vacuole (Odorizzi et al., 1998). GFP-CPS was found en- of Doa4 is essential for its role as a suppressor, indicating tirely within the vacuole lumen of wild-type cells (Fig. 1 B). CC that the mechanism of suppression involves substrate deu- In contrast, GFP-CPS in bro1 cells was observed at the biquitination. Furthermore, the multivesicular morphology vacuole membrane (Fig. 1 B), which is indicative of a defect of late endosomes, which is abnormal in bro1 mutant cells, in sorting of the fusion protein via the MVB pathway is restored by the overexpression of DOA4. The ability of (Odorizzi et al., 1998). GFP-CPS was also mislocalized to high-copy DOA4 to suppress the loss of Bro1 function is class E compartments (Fig. 1 B, arrowheads), abnormal late explained by our finding that Bro1 is essential for the local- endosomal structures that occur in all class E vps mutant ization of Doa4 to endosomes and that Bro1 and Doa4 cells. A similar defect in the localization of GFP-CPS was physically interact on endosomal membranes. Therefore, seen in bro1 cells in which the entire BRO1 coding se- Bro1 has a crucial role in coordinating substrate deubiquiti- quence had been deleted (Fig. 1 B). In addition, we observed CC nation in the MVB pathway by recruiting Doa4 to endo- in both bro1 and bro1 mutant cells that Ste3-GFP, an somes. MVB cargo protein endocytosed from the plasma mem- brane (Urbanowski and Piper, 2001), failed to be sorted into the vacuole lumen (unpublished data). Thus, the coiled-coil domain of Bro1 is essential for the function of Bro1 in the Results MVB pathway. CC The coiled-coil domain of Bro1 is essential for Interestingly, when we overexpressed the bro1 mutant MVB sorting allele in wild-type cells, GFP-CPS was mislocalized both to Many class E Vps proteins have one or more coiled-coil do- the vacuole membrane and to a structure that resembles the mains, which are structural elements that typically mediate class E compartment (Fig. 1 C). In contrast, overexpression protein–protein interactions. Bro1 has a central coiled-coil of the wild-type BRO1 gene had no deleterious effect on the domain spanning residues 543-583 (Fig. 1 A). We con- localization of GFP-CPS (Fig. 1 C). The overexpression of CC CC structed a mutant allele (bro1 ) in which the sequence en- bro1 , therefore, causes a dominant-negative phenotype, coding this domain had been deleted, and then integrated possibly because the mutant gene product interferes with the CC the bro1 allele by homologous recombination into the ge- function of the wild-type Bro1 protein. Bro1 recruits Doa4 to endosomes | Luhtala and Odorizzi 719 GFP, fluorescence was localized at class E compartments that were costained by FM 4-64 (Fig. 2, C and D, closed ar- rowheads). Class E compartments had formed in VPS4 CC cells expressing bro1 -GFP in place of wild-type BRO1 due to the fact that the coiled-coil domain is required for the function of Bro1 in the MVB pathway (Fig. 1). Electron mi- croscopic analysis of immunogold labeled cell sections con- CC firmed that the bro1 -GFP fusion protein was, indeed, as- sociated with class E compartments in both VPS4 and vps4 cells (not depicted). Interestingly, in contrast with CC wild-type Bro1-GFP, bro1 -GFP was also localized in both strains to additional punctate structures that were not labeled by FM 4-64 (Fig. 2, C and D, open arrowheads). CC However, the localization of bro1 -GFP to class E com- partments indicates that the coiled-coil domain per se is not required for the localization of Bro1 to late endosomes. Overexpression of DOA4 suppresses CPY sorting defects in bro1 mutant cells To identify proteins that may be able to restore normal function of the MVB pathway when over-produced in the CC bro1 mutant strain, we designed a genetic screen based upon another phenotype observed in bro1 mutant cells. In addition to MVB sorting defects, bro1 mutants inefficiently sort a soluble vacuolar enzyme, carboxypeptidase Y (CPY). Newly synthesized CPY is transported from the ER to the Golgi, where it binds a transmembrane receptor, Vps10. In wild-type cells (Fig. 3 A), the Vps10–CPY complex is sorted from the Golgi to endosomes, whereupon CPY dissociates Figure 2. The Bro1 coiled-coil domain is not required for endosomal from Vps10, and the receptor recycles back to the Golgi, localization of Bro1. Fluorescence and DIC microscopy of DMY1 whereas CPY is transported further toward the vacuole (Cere- (A), DMY2 (B), DBY15 (C), and DBY18 (D) cells stained with FM ghino et al., 1995; Cooper and Stevens, 1996). The class E 4-64. Closed arrowheads indicate class E compartments costained compartments that are formed in bro1 mutant cells prevent by FM 4-64. Open arrowheads indicate GFP-positive structures not labeled by FM 4-64. Bars, 2.5 m. Vps10 from efficiently recycling to the Golgi (Fig. 3 B), re- sulting in a significant portion of newly synthesized CPY en- tering by default into the secretory pathway (Odorizzi et al., The coiled-coil domain is not required for Bro1 to 2003). Thus, the secretion of CPY by bro1 mutant cells is associate with endosomes likely to be an indirect consequence of the aberrant endoso- We previously demonstrated that Bro1 is a cytoplasmic pro- mal morphology that occurs upon the loss of Bro1 function. tein which associates with endosomal compartments and that The percentage of total cellular CPY that is secreted can be the dissociation of Bro1 from endosomes requires the ATPase measured quantitatively using a colorimetric assay that moni- activity of Vps4 (Odorizzi et al., 2003). Thus, when we re- tors the enzymatic activity of a CPY-invertase (CPY-Inv) fu- placed the wild-type BRO1 gene with a genomically inte- sion protein (Darsow et al., 2000). As shown in Fig. 3 C, grated BRO1-GFP gene fusion in cells that have normal Vps4 wild-type cells secreted 1% of CPY-Inv, whereas bro1 CC function (VPS4 ), we observed multiple fluorescent punctate cells secreted 10–15 times this amount. The bro1 mutant structures in addition to diffuse cytoplasmic fluorescence (Fig. strain secreted nearly the same percentage as bro1 cells (Fig. 2 A). Upon deletion of the VPS4 gene (vps4), the GFP fluo- 3 C), which is consistent with the coiled-coil domain being rescence was concentrated at class E compartments, which essential for the function of Bro1 in the MVB pathway. had formed in these cells due to the absence of Vps4 activity The colorimetric assay for invertase activity can also be (Fig. 2 B). The cells in these experiments shown in Fig. 2 (and used to detect the secretion of CPY-Inv by cells growing on in Figs. 6, 7, and 9) were also labeled by a brief pulse of FM solid medium (Darsow et al., 2000). We used this assay in a 4-64 followed by incubation in label-free medium. FM 4-64 genetic screen to identify any gene that, when overexpressed CC is a fluorescent lipophilic compound that intercalates into the in bro1 cells, reduced the amount of secreted CPY-Inv. plasma membrane and is endocytosed, resulting in the stain- We reasoned that such a high-copy suppressor would encode ing of vacuole membranes and class E compartments (Vida a protein that interacts with Bro1 and would be able to re- and Emr, 1995). store normal function of the MVB pathway when over pro- CC To determine if the coiled-coil domain is required for duced. Thus, we transformed the bro1 strain with a high- the association of Bro1 with endosomes, we integrated a copy plasmid library of yeast genomic DNA and identified CC CC bro1 -GFP gene fusion in place of the wild-type BRO1 colonies that secreted less CPY-Inv relative to the bro1 CC gene. Both in VPS4 and vps4 cells expressing bro1 - strain. One clone contained a plasmid that could reproduc- 720 The Journal of Cell Biology | Volume 166, Number 5, 2004 mately twofold the amount of CPY-Inv secreted by bro1 cells, indicating a partial bypass of Bro1. The catalytic region of UBP enzymes have a conserved three-domain architecture that includes a critical cysteine residue, which forms a thiolester bond with Ub (Hu et al., C/S 2002). We constructed a point mutant allele (doa4 ) in which the corresponding cysteine in Doa4 (Cys ) had been C/S changed to a serine residue. The doa4 allele failed to suppress CPY-Inv secretion when overexpressed in either CC C/S bro1 or bro1 cells. Instead, doa4 overexpression en- hanced the mutant phenotype in both strains and caused a stronger dominant-negative phenotype in wild-type cells (Fig. 3 E). Catalytic activity, therefore, is essential for high- copy DOA4 to suppress CPY-Inv secretion caused by the loss of Bro1 function. Because high-copy DOA4 partially suppressed the bro1 phenotype, we determined whether the loss of any class E Vps protein could be bypassed by DOA4 overexpression. We transformed either the empty library vector or the library plasmid containing DOA4 into strains in which individual class E VPS genes had been deleted, and then measured the percentage of secreted CPY-Inv (Table I). Suppression failed to occur in strains in which components of the ESCRT-I, -II, or -III complexes had been deleted (Table I). High-copy DOA4 also could not suppress the loss of other class E Vps proteins, including Vps27 (Table I), which functions upstream of the ESCRT-I complex in the recognition of ubiquitinated MVB cargo proteins (Bilodeau et al., 2003; Katzmann et al., 2003). Interestingly, DOA4 overexpression suppressed the secretion of CPY-Inv by vps4 cells but could Figure 3. DOA4 overexpression in bro1 mutant cells suppresses CC CPY-Inv secretion. Schematic diagram of the trafficking of CPY in not suppress either bro1 vps4 or bro1 vps4 double wild-type cells (A) and in bro1 mutant cells (B). Not depicted in A mutant cells (Table I). These observations suggest that high- is the fusion of the endosome/MVB with the vacuole. PM, plasma copy DOA4 suppresses the CPY sorting defects in bro1 and membrane. (C–E) Quantitation of CPY-Inv secretion by BHY10, vps4 mutants by different mechanisms. GOY57, and KGY1 cells transformed either with the empty high- copy (2 ) plasmid (C), the 2  plasmid containing the wild-type C/S High-copy DOA4 suppresses CPS deubiquitination and DOA4 gene (D), or the 2  plasmid containing the mutant doa4 CC allele (E). The bar graph depicts the mean  standard error from sorting defects in bro1 mutant cells multiple independent experiments (Table I). We had hypothesized that a high-copy suppressor of the ab- errant secretion of CPY-Inv by bro1 mutant cells would re- store normal function of the MVB pathway. Indeed, when ibly suppress CPY-Inv secretion. Nucleotide sequence analy- CC sis revealed that this suppressor plasmid contained a 7.3-kb we examined the localization of GFP-CPS in bro1 cells overexpressing DOA4, most of the fusion protein was seen fragment of chromosome IV that includes the complete ORF of five genes, one of which is DOA4. inside the vacuole lumen, with only a small amount detected on the vacuole membrane (Fig. 4 A). Fluorescent punctate The DOA4 gene product is a member of the Ub-specific processing protease (UBP) family of Ub thiolesterases (Wing, structures were not evident in these cells, suggesting that class E compartment formation was significantly reduced. 2003). Doa4 is the UBP required for removal of Ub from the cytoplasmic domain of CPS and other MVB cargo pro- We also observed more GFP-CPS within the vacuole lumen of bro1 cells overexpressing DOA4 (Fig. 4 B) compared teins (Dupre and Haguenauer-Tsapis, 2001; Katzmann et al., 2001; Losko et al., 2001), and subcloning confirmed with bro1 cells alone (Fig. 1 B). However, compared with CC bro1 cells that overexpress DOA4 (Fig. 4 A), much more that DOA4 is the gene within the library plasmid responsible CC for suppression of the bro1 phenotype (unpublished GFP-CPS in bro1 cells overexpressing DOA4 was seen at the vacuole membrane and at structures that resembled class data). The suppression of CPY-Inv secretion mediated by DOA4 overexpression is shown in Fig. 3 D and Table I. High- E compartments (Fig. 4 B). Thus, high-copy DOA4 is much less efficient at suppressing the defect in GFP-CPS sorting in copy DOA4 reduced the percentage of CPY-Inv secreted by CC CC bro1 cells by approximately threefold. In contrast, DOA4 bro1 compared with bro1 cells. Although the overexpression of DOA4 in vps4 cells sup- overexpression resulted in approximately threefold more CPY-Inv secreted by wild-type cells, indicating that high- pressed CPY-Inv secretion (Table I), it did not restore the sorting of GFP-CPS into the vacuole lumen. As shown in copy DOA4 caused a subtle dominant-negative phenotype. Surprisingly, DOA4 overexpression reduced by approxi- Fig. 4 D, GFP-CPS was mislocalized to the vacuole mem- Bro1 recruits Doa4 to endosomes | Luhtala and Odorizzi 721 Table I. Effect of DOA4 over-expression on CPY-Inv secretion and GFP-CPS sorting / 2 µ Percentage of a b Strain DOA4 secreted CPY-Inv GFP-CPS localization c d e 1.0  2.1 (5) VL Wild-type 2.8  1.5 (4) VL 11.5  4.7 (24) VM  EC ∆ CC bro1 3.7  2.5 (7) VL 13.0  3.8 (6) VM  EC bro1∆ 5.6  2.4 (5) VL  VM  EC 39.4  2.5 (3) VM  EC vps4∆ 5.2  1.7 (3) VM  EC 43.7  6.9 (3) VM  EC bro1∆ vps4∆ 46.0  7.1 (3 VM  EC 35.2  2.9 (3) VM  EC ∆ CC bro1 vps4∆ 35.9  4.1 (3) VM  EC ESCRT-I mutants 20.0  5.9 (3) VM  EC vps23∆ 17.6  2.0 (3) VM  EC 13.4  1.2 (3) VM  EC vps28∆ 21.7  4.2 (3) VM  EC 16.3  1.7 (3) VL vps37∆ 15.8  1.3 (3) VL ESCRT-II mutants 20.1  4.5 (3) VM  EC vps22∆ 20.5  6.1 (3) VM  EC 23.1  1.0 (3) VM  EC vps25∆ 20.7  0.9 (3) VM  EC 24.2  6.3 (3) VM  EC vps36∆ 20.6  4.1 (3) VM  EC ESCRT-III mutants 25.2  5.0 (3) VM  EC vps2∆ 27.8  6.5 (3) VM  EC 29.1  4.3 (3) VM  EC vps24∆ 30.9  4.4 (3) VM  EC 24.2  2.9 (3) VM  EC vps20∆ 20.1  6.5 (3) VM  EC 25.8  5.0 (3) VM  EC snf7∆ 24.6  1.1 (3) VM  EC Other class E vps mutants tested 28.0  1.5 (3) VM  EC vps27∆ 26.5  2.6 (3) VM  EC 41.8  13.0 (3) VL nhx1∆ 39.1  13.0 (3) VL vps60∆  17.7  1.3 (3) VM  EC 19.0  5.5 (3) VM  EC Strains were transformed with the empty 2 µ vector () or the 2 µ vector containing DOA4 (). The percentage of total cellular CPY-Inv secreted by cells. Mean  standard error. Number of independent experiments. Vacuole lumen. Vacuole membrane and class E compartment. There was no noticeable defect in GFP-CPS sorting in vps37 and nhx1 cells. brane and the class E compartment in vps4 cells overexpress- Bro1 function. Because the deubiquitination of CPS is nor- ing DOA4, which is identical to the mislocalization of GFP- mally efficient, Ub-CPS was difficult to detect in wild-type CPS in vps4 cells (Odorizzi et al., 1998). High-copy DOA4 cells, but a significant amount of Ub-CPS was observed in CC also failed to restore the sorting of GFP-CPS via the MVB bro1 cells (Fig. 4 E). As shown in Fig. 4 (E and F), overex- pathway in other class E vps mutants (Table I). pression of DOA4 completely restored CPS deubiquitination CC In doa4 mutant cells, CPS and other MVB cargoes accu- in bro1 cells, which is consistent with a role for Doa4 in mulate in their ubiquitinated forms (Dupre and Haguenauer- deubiquitinating MVB cargo proteins. Tsapis, 2001; Katzmann et al., 2001; Losko et al., 2001). We Interestingly, much more Ub-CPS was observed in bro1 CC recently showed that ubiquitinated CPS (Ub-CPS) also accu- cells compared with the level of Ub-CPS seen in bro1 mulates in bro1 cells (Odorizzi et al., 2003). Therefore, we cells (Fig. 4 E), indicating that the deubiquitination of CPS investigated whether DOA4 overexpression could alleviate is more severely compromised if the Bro1 protein is absent. the defect in CPS deubiquitination that occurs upon loss of Furthermore, the amount of Ub-CPS in bro1 mutants 722 The Journal of Cell Biology | Volume 166, Number 5, 2004 Figure 5. DOA4 overexpression restores MVB morphology in bro1 CC mutant cells. (A) EM showing MVBs in BHY10 cells. Arrowheads Figure 4. DOA4 overexpression in bro1 mutant cells restores indicate MVBs. n, nucleus. Bar, 0.2 m. (B) MVBs from the boxed the sorting and deubiquitination of CPS. (A–D) Fluorescence and area shown in A. Bar, 0.1 m. (C) EM showing a typical class E DIC microscopy of GOY57 (A), KGY1 (B), BHY10 (C), and DBY42 compartment in GOY57 cells. Bar, 0.1 m. (D) EM showing multi- (D) cells expressing the GFP-CPS fusion and transformed with 2 vesicular structures in GOY57 cells transformed with 2  DOA4. DOA4. Arrowheads indicate class compartments. Bars, 2.5 m. The arrowhead indicates a tubule projecting from an MVB toward (E) Immunoprecipitates of CPS were examined by Western blotting the cytoplasm; note that the membrane bilayer of the tubule is using anti-Ub or anti-CPS antibodies. Note that the lumenal domain continuous with the bilayer surrounding the lumenal vesicles. Bar, of CPS is differentially glycosylated and is observed as a doublet. 0.1 m. (E) MVBs and class E compartments were counted in 200 (F) Quantitation of the intensities of Ub-CPS versus CPS present cells which had a diameter  2.5 m. Each value is indicated above in each lane in E. Bar graph depicts mean values  SD from two its respective bar in the graph and was expressed as the number per independent experiments. 200 cells. The multivesicular structures that had tubular projections (D, arrowhead) were counted as MVBs. was much less significantly reduced upon overexpression of DOA4, in contrast with the complete restoration of CPS CC High-copy DOA4 restores MVB morphology in deubiquitination that occurred in bro1 cells. This result bro1 mutant cells is consistent with our observation that high-copy DOA4 was much more effective at suppressing the GFP-CPS sort- By fluorescence microscopy, it appeared that the overexpres- CC ing defect in bro1 cells (Fig. 4 A) versus bro1 cells (Fig. sion of DOA4 caused a reduction in the number of class E 4 B). Thus, excess amounts of Doa4 effectively alleviate the compartments in bro1 mutant cells. Therefore, we used EM mutant phenotype caused by deletion of the Bro1 coiled- to examine the effect that high-copy DOA4 has on endoso- coil domain but cannot substitute for deletion of the entire mal ultrastructure. In wild-type cells, we readily observed Bro1 protein. MVBs, with each compartment consisting of a limiting Bro1 recruits Doa4 to endosomes | Luhtala and Odorizzi 723 tures, examples of which are shown in Fig. 5 D. These com- partments consisted of a limiting membrane bilayer that en- circled multiple vesicular profiles within the compartment lumen, which is similar to MVBs in wild-type cells (Fig. 5 B). Quantitative analysis indicated that DOA4 overexpres- CC sion in bro1 cells caused a dramatic reduction in the number of class E compartments and resulted in almost as many MVBs as we had observed in wild-type cells (Fig. 5 E). The apparent reformation of MVBs and the concomitant disappearance of class E compartments upon DOA4 overex- pression occurred to a lesser extent in bro1 cells (Fig. 5 E), again indicating that high-copy DOA4 is not as efficient at suppressing the phenotype caused by a total loss of Bro1 compared with its ability to suppress the phenotype caused by a deletion of only the Bro1 coiled-coil domain. Interestingly, the overexpression of DOA4 in bro1 mutant cells occasionally resulted in multivesicular compartments having tubules projecting toward the cytoplasm that resemble the cisterna-like structures of class E compartments (Fig. 5 D, arrowhead). Although further studies are needed in order to establish the identity of these unusual structures, they may correspond to intermediate MVB/class E compartments. The localization of Doa4 to endosomes requires Bro1 Doa4 had previously been shown to associate with endoso- mal compartments (Amerik et al., 2000). Indeed, when we integrated a DOA4-GFP gene fusion in place of the wild- type DOA4 gene in cells that have normal Vps4 function (VPS4 ), we observed multiple fluorescent punctate struc- tures in addition to diffuse cytoplasmic fluorescence (Fig. 6 A). This pattern of fluorescence resembles the intracellular localization of Bro1-GFP in VPS4 cells (Fig. 2 A). Also like Bro1-GFP, Doa4-GFP was concentrated at class E compart- ments in vps4 cells (Fig. 6 B). Doa4-GFP was also localized CC CC to class E compartments in bro1 and bro1 vps4 cells (Fig. 6, C and D, closed arrowheads), indicating that the Bro1 coiled-coil domain is not required for the association of Doa4 with late endosomes. Interestingly, Doa4-GFP was localized to additional punctate structures that were not la- beled by FM 4-64 in VPS4 and vps4 cells expressing the CC mutant bro1 allele in place of the wild-type BRO1 gene (Fig. 6, C and D, open arrowheads), which is similar to the CC localization pattern of the mutant bro1 -GFP protein (Fig. Figure 6. Bro1 is required for the localization of Doa4 to endosomes. 2, C and D). Fluorescence and DIC microscopy of GOY74 (A), GOY75 (B), GOY88 (C), GOY93 (D), GOY82 (E), and GOY83 (F) cells stained A dramatic change in the localization of Doa4-GFP oc- with FM 4-64. Closed arrowheads indicate colocalization of GFP curred in cells in which the BRO1 gene had been deleted. and FM 4-64 at class E compartments. In C and D, open arrowheads Both in bro1 cells (Fig. 6 E) and bro1 vps4 double mu- indicate GFP-positive structures not labeled by FM 4-64. In E and F, tant cells (Fig. 6 F), Doa4-GFP failed to localize to class E open arrowheads indicate FM 4-64–positive class E compartments compartments and was, instead, diffusely distributed. Some at which Doa4-GFP is not localized. Bars, 2.5 m. faintly fluorescent structures were evident at the periphery of these cells but are unlikely to be endosomes, as they were membrane bilayer surrounding numerous vesicular profiles never stained when FM 4-64 was incubated continuously (Fig. 5, A and B). In contrast, MVBs were never evident in with cells (unpublished data), a procedure that results in CC bro1 or bro1 cells, which, instead, contained numerous staining of all compartments of the endocytic pathway (Vida class E compartments. An example of a class E compartment and Emr, 1995). Western blot analysis confirmed that the CC seen in bro1 cells is shown in Fig. 5 C. Similar structures expression of full-length Doa4-GFP in bro1 and bro1 were also seen in bro1 cells (not depicted). vps4 double mutant cells was equivalent to its expression High-copy DOA4 caused a striking change in endosomal in wild-type and vps4 cells (Fig. S1, available at http:// CC morphology in bro1 mutant cells. Rather than class E www.jcb.org/cgi/content/full/jcb.200403139/DC1), indicat- compartments, we observed numerous multivesicular struc- ing that the loss of punctate localization of Doa4-GFP in the 724 The Journal of Cell Biology | Volume 166, Number 5, 2004 Figure 7. Doa4 is not required for the localization of Bro1 to endosomes. Fluorescence and DIC microscopy of GOY69 (A) and GOY70 (B) cells stained with FM 4-64. Arrowheads indicate class E compartments. Bars, 2.5 m. absence of Bro1 was not due to aberrant cleavage of the fu- sion protein. Furthermore, transformation of a plasmid en- CC Figure 8. Doa4 interacts with Bro1 and the mutant Bro1 protein coding the wild-type BRO1 gene into bro1 and bro1 on membranes. (A) Detergent extracts of total lysates from GOY107, vps4 cells restored the localization of Doa4-GFP to endo- GOY108, GOY106, and GOY105 cells were subjected to immuno- somal compartments (unpublished data). Thus, Bro1 is es- precipitation with rabbit anti-GFP polyclonal antiserum under native sential for the localization of Doa4 to endosomes. conditions and examined by Western blotting. (B) Cell lysates were separated into membrane pellet (P) and soluble (S) fractions and To test whether the endosomal localization of Bro1 is like- extracted with detergent before immunoprecipitation and Western wise dependent on Doa4, we examined the localization of blot analysis. Bro1-GFP in cells in which the DOA4 gene had been de- leted. In doa4 VPS4 cells, we observed punctate fluores- CC Bro1-HA and bro1 -HA coimmunoprecipitated with Doa4- cent structures in addition to diffuse cytoplasmic fluores- GFP from the membrane fraction of VPS4 cells; however, cence (Fig. 7 A), whereas in doa4 vps4 double mutant these interactions were more significantly enriched in mem- cells, the GFP fluorescence was concentrated at class E com- brane extracts considering that the vast majority of Doa4- partments (Fig. 7 B). This pattern of fluorescence was virtu- GFP in VPS4 cells was recovered from the soluble fraction ally identical to the intracellular localization of Bro1-GFP in (Fig. 8 B). In vps4 cells, the interaction between Doa4- VPS4 and vps4 cells (Fig. 2). Therefore, Doa4 is not re- CC GFP and both Bro1-HA and bro1 -HA occurred almost quired for the endosomal localization of Bro1. exclusively in the membrane fraction, again despite the fact that more Doa4-GFP was immunoprecipitated from the sol- Doa4 interacts with Bro1 uble fraction (Fig. 8 B). Altogether, these results indicate Because Bro1 is required for the endosomal localization of that Doa4 associates with Bro1 on membranes and that the Doa4, we investigated whether Doa4 physically interacts coiled-coil domain is not required for this interaction to with Bro1 by immunoprecipitating the Doa4-GFP fusion occur. protein from detergent-solubilized extracts of yeast cell lysates under native conditions. As shown in Fig. 8 A, CC Endosomal localization of Doa4 in ESCRT-III Bro1-HA and bro1 -HA fusion proteins coimmunopre- mutant cells cipitated with Doa4-GFP from total extracts of VPS4 and vps4 cells. Control immunoprecipitations from lysates of A previous study had suggested that the association of Doa4 strains not expressing DOA4-GFP confirmed that the isola- with endosomes was dependent on two other class E Vps pro- tion of Bro1-HA using anti-GFP antiserum required Doa4 teins, Vps24 and Snf7 (Amerik et al., 2000). Both Vps24 and (Fig. S2, available at http://www.jcb.org/cgi/content/full/ Snf7 are components of the ESCRT-III complex that oligo- jcb.200403139/DC1). Furthermore, the interaction between merizes on endosomal membranes (Babst et al., 2002a). We Bro1-HA and Doa4-GFP also occurred in anti-HA immu- recently showed that the endosomal localization of Bro1 re- noprecipitations followed by anti-GFP Western blots (un- quires Snf7 but not Vps24, suggesting that Snf7 has a role in published data). the recruitment or stabilization of Bro1 at endosomes (Odo- We tested whether the interaction between Doa4 and rizzi et al., 2003). Therefore, we examined the localization of Bro1 occurred on membranes and/or in the cytoplasm by Doa4-GFP in vps4 cells in which either VPS24 or SNF7 centrifuging cell lysates before immunoprecipitation in or- had been deleted. In contrast with the previous analysis of der to generate a pellet fraction enriched in late endosomal Doa4 localization (Amerik et al., 2000), we observed Doa4- membranes and a supernatant fraction containing soluble GFP at class E compartments in vps24 vps4 double mu- proteins (Odorizzi et al., 2003). As shown in Fig. 8 B, more tant cells (Fig. 9 A). However, Doa4-GFP was diffusely dis- Bro1 recruits Doa4 to endosomes | Luhtala and Odorizzi 725 Figure 10. Model for the recruitment of Doa4 to endosomes by Bro1. Bro1 is required for the association of Doa4 with endosomes, where Doa4 catalyzes deubiquitination of MVB cargo proteins. The endosomal recruitment of Bro1, and consequently the recruit- ment of Doa4, is dependent on Snf7, which oligomerizes on endosomal membranes with the other ESCRT-III components (Vps2, Vps24, and Vps20). After their deubiquitination, MVB cargoes are sorted into lumenal vesicles. Not depicted is the dissociation of Bro1, Doa4, and ESCRT-III proteins from endosomes, which is catalyzed by the Vps4 ATPase. Recruitment of Doa4 by Bro1 Both Doa4-GFP and Bro1-GFP were associated with endoso- mal compartments, but upon deletion of the BRO1 gene, the localization of Doa4-GFP was shifted to the cytoplasm. In contrast, Bro1-GFP remained associated with endosomes re- gardless of whether the DOA4 gene had been deleted. Native Figure 9. Localization of Doa4-GFP in ESCRT-III mutants. (A and B) immunoprecipitation experiments revealed a physical interac- Fluorescence and DIC microscopy of GOY113 (A) and GOY114 (B) tion between Doa4 and Bro1, which could reflect direct bind- cells stained with FM 4-64. Closed arrowheads indicate colocalization ing of Doa4 by Bro1 or, alternatively, could be mediated by of Doa4-GFP and FM 4-64 at class E compartments. Open arrow- heads indicate class E compartments at which Doa4-GFP is not another component that binds both proteins. In either case, localized. Bars, 2.5 m. (C) Native immunoprecipitations from the association of Doa4 with Bro1 occurred predominantly in detergent extracts of total lysates of GOY115 and GOY116 cells. subcellular fractions containing late endosomal membranes, which is consistent with the ordered recruitment of Bro1 to endosomes followed by the recruitment of Doa4 (Fig. 10). tributed in snf7 vps4 double mutants (Fig. 9 B). This pat- tern in the localization of Doa4-GFP was identical to the Consistent with this model, the association of Doa4-GFP with endosomes required Snf7, a component of the ESCRT- localization of Bro1 that we had previously observed in these same mutant strains (Odorizzi et al., 2003). The inability of III complex. We recently showed that Snf7 is essential for the endosomal localization of Bro1 (Odorizzi et al., 2003), and Doa4-GFP to localize to endosomal compartments in the ab- sence of Snf7 may, therefore, be due to the requirement for in mammalian cells, the orthologue of Bro1, Alix/Aip1, binds Chmp4b, which is an orthologue of Snf7 (Katoh et al., Snf7 in the endosomal localization of Bro1 (Odorizzi et al., 2003). Indeed, as shown in Fig. 9 C, neither Vps24 nor Snf7 2003; Martin-Serrano et al., 2003; Strack et al., 2003; von Schwedler et al., 2003; Peck et al., 2004). Thus, it is likely coimmunoprecipitated with Doa4 from VPS4 or vps4 cell extracts. Thus, the ESCRT-III complex does not appear to that Snf7 has an indirect role in the endosomal localization of Doa4 by functioning to recruit Bro1 to endosomes or to sta- have a direct role in the recruitment of Doa4 to endosomes. bilize the association of Bro1 with endosomal membranes. In- deed, we were unable to detect a physical interaction between Discussion Doa4 and either Snf7 or Vps24, another component of the Protein sorting in the MVB pathway requires Bro1, a cyto- ESCRT-III complex. Furthermore, high-copy DOA4 could plasmic protein that associates with endosomal compart- not suppress the defects in either CPY or CPS sorting caused ments (Odorizzi et al., 2003). Here, we show that DOA4 by the loss of any ESCRT-III component. overexpression suppresses the defects in MVB transport and It is unlikely that Snf7 recruits Bro1 to endosomes by in- restores the multivesicular morphology of endosomes in teracting with the Bro1 coiled-coil domain, as this region of bro1 mutant cells. These observations can be explained by Bro1 was not required for its association with endosomal our finding that Bro1 associates with Doa4 on endosomal membranes. Furthermore, despite the fact that we identified CC membranes and that the recruitment of Doa4 to endosomes DOA4 in a screen for high-copy suppressors of the bro1 requires Bro1. Thus, our results indicate a key role for Bro1 mutant phenotype, the Bro1 coiled-coil domain is not nec- in regulating the timing and location of Doa4 activity in the essary for Bro1 to interact with Doa4. Nevertheless, the MVB pathway. coiled-coil domain is important for Bro1 function, as the 726 The Journal of Cell Biology | Volume 166, Number 5, 2004 CC transport of GFP-CPS was blocked in bro1 mutant cells Consistent with a role for Bro1 in coordinating Doa4 as effectively as it was in bro1 cells. Therefore, the recruit- function are the previous observations that Bro1 is re- ment of Doa4 to endosomes is only one aspect of the func- quired for the deubiquitination of CPS (Odorizzi et al., tion of Bro1 in the MVB pathway. The role of the Bro1 2003) as well as Gap1, an amino acid permease at the coiled-coil domain is not yet clear. Thus far, we have been plasma membrane that is down-regulated by endocytosis unable to identify another protein that interacts with this re- and sorted via the MVB pathway (Nikko et al., 2003). In- gion of Bro1, and we have been unable to detect homo-oli- terestingly, in bro1 mutant cells, Gap1 is recycled to the gomerization of Bro1 that could be mediated by the coiled- plasma membrane but, unlike CPS, does not accumulate coil domain (unpublished data). in its ubiquitinated form. However, ubiquitinated Gap1 is readily detected in bro1 mutant cells if recycling is blocked, Suppression by high-copy DOA4 suggesting that in the absence of Bro1, the deubiquitina- The restoration of multivesicular structures upon the overex- tion of Gap1 (and possibly other endocytic cargoes) occurs pression of DOA4 coincided with the disappearance of class elsewhere within the cell and may be catalyzed by one of E compartments, which presumably enables the CPY recep- the 15 other UBPs in yeast (Nikko et al., 2003). tor, Vps10, to recycle to the Golgi where it binds newly syn- The suppressor activity of high-copy DOA4 was abol- thesized CPY-Inv in order to transport this soluble cargo ished if the putative active-site cysteine residue was altered protein to the endosome (Cereghino et al., 1995; Cooper to serine, indicating that suppression most likely occurred and Stevens, 1996). Accordingly, we have observed that a through substrate deubiquitination. However, the deubiq- Vps10-GFP fusion protein was not concentrated at class E uitination of cargoes is not essential for MVB sorting be- compartments in bro1 mutant cells overexpressing DOA4 cause chimeric cargo proteins that are expressed as transla- but was, instead, localized to multiple punctate structures tional fusions to Ub cannot be deubiquitinated yet are (unpublished data), which is similar to its localization in transported efficiently via the MVB pathway (Reggiori and wild-type cells (Burda et al., 2002; Odorizzi et al., 2003). Pelham, 2001; Urbanowski and Piper, 2001; Bilodeau et Interestingly, although high-copy DOA4 restored the mul- al., 2003). Nevertheless, Doa4 is required for the MVB tivesicular morphology of late endosomes and suppressed pathway, as the sorting of MVB cargo proteins is blocked CC CPY-Inv secretion by both bro1 and bro1 cells, the in doa4 mutant cells (Losko et al., 2001; Reggiori and Pel- transport of GFP-CPS via the MVB pathway was efficiently ham, 2001). In addition to MVB cargoes, Doa4 could CC restored by DOA4 overexpression only in bro1 and not deubiquitinate a component of the MVB sorting machin- bro1 cells. Furthermore, the overexpression of DOA4 in ery in order to regulate its activity. It is not known whether bro1 cells did not significantly enhance the deubiquitina- class E Vps proteins in yeast are regulated by ubiquitina- tion of CPS, whereas the minor amount of Ub-CPS seen in tion. In mammalian cells, however, several cytoplasmic CC the bro1 strain was completely eliminated by DOA4 proteins that control receptor down-regulation are mono- overexpression. These observations are consistent with the ubiquitinated, including Hrs, which is the orthologue of ability of Doa4 to be recruited to endosomes in cells express- the yeast class E Vps protein, Vps27 (Polo et al., 2002). CC ing the mutant bro1 protein but not in cells in which CIN85 and endophilin are also monoubiquitinated cy- Bro1 is completely absent. Moreover, these findings suggest toplasmic components in mammalian cells that associate that restoring MVB morphology alone by DOA4 overex- with endosomes (Haglund et al., 2002; Angers et al., pression is not sufficient for a complete restoration of the 2004), and both proteins interact with Alix/Aip1 (Vito et MVB pathway. The overexpression of DOA4 in bro1 cells al., 1996; Chatellard-Causse et al., 2002). Like Bro1 in may result in excess amounts of Doa4 that could deubiquiti- yeast, Alix/Aip1 could recruit a UBP that deubiquitinates nate proteins which regulate the formation of MVB vesicles, CIN85 and endophilin in order to regulate their function but the sorting of cargoes into MVB vesicles per se may be and/or localization. critically dependent on the coordination of Doa4 on endo- Our results suggest a model in which Bro1 recruits Doa4 somal membranes by Bro1 as well as other aspects of Bro1 to endosomal membranes, thereby controlling the timing function in the MVB pathway. and location of Doa4 activity in the MVB pathway (Fig. 10). The association of Bro1 itself with endosomes occurs Deubiquitination in the MVB pathway after the assembly of the ESCRT-III complex on endo- Ubiquitination is tightly controlled by Ub-conjugation en- somes (Odorizzi et al., 2003). Similarly, the deubiquitina- zymes and is counterbalanced by deubiquitination. Both tion of MVB cargo proteins occurs downstream of the poly- and monoubiquitinated proteins undergo deubiqui- functions of the ESCRT-I, -II, and -III complexes (Nikko tination, but few Ub thiolesterases have been assigned to et al., 2003) but before enclosure of cargoes within MVB specific substrates (Wing, 2003). There are 16 UBPs in S. vesicles (Hicke and Dunn, 2003). Thus, the Bro1-depen- cerevisiae, suggesting that many of these enzymes have dent coordination of Doa4 on endosomal membranes and highly specific and regulated functions. However, Doa4 the subsequent deubiquitination of cargo proteins (and probably has a wide variety of substrates. In addition to its possibly MVB sorting components) is likely to be one of role in the removal of monoubiquitin from MVB cargo the last steps in the MVB pathway, ensuring that cargo proteins, Doa4 is likely to function in the removal of poly- proteins are concentrated at regions of the endosomal ubiquitin from proteasomal substrates, as Doa4 copurifies membrane where invagination occurs. In mammalian cells, with proteasomes that have been isolated from yeast cell the assembly/budding of HIV-1 depends on ubiquitina- extracts (Papa et al., 1999). tion of the viral Gag protein (for review see Pornillos et al., Bro1 recruits Doa4 to endosomes | Luhtala and Odorizzi 727 CC allele, the SpeI–SalI fragment containing the To construct the bro1 2002), but a role for deubiquitination in this process has BRO1 gene in plasmid pGO187 (Odorizzi et al., 2003) was subcloned not been described. However, recent studies indicate that into SpeI–SalI-digested pRS413, resulting in plasmid pGO263, which Alix/Aip1 interacts with both the ESCRT-I and -III com- was digested with EcoRV and recircularized using T4 DNA ligase. The CC allele consist- plexes and is involved in viral escape from host cells (Strack resulting plasmid (pGO265) contained the mutant bro1 ing of codon 540 adjoined to codon 586. The SpeI–SalI fragment in et al., 2003; von Schwedler et al., 2003). Future studies pGO265 was subcloned into SpeI–SalI-digested pRS416, pRS426, and may determine whether Alix/Aip1 has a role analogous to pRS306, resulting in pGO273, pGO272, and pGO287, respectively. The CC Bro1 and functions in the recruitment of a specific UBP to allele was integrated into the genome in place of the wild-type bro1 BRO1 gene using pGO287 by homologous recombination (Guthrie and the site of viral budding. C/S allele was constructed by PCR and subcloned Fink, 2002). The doa4 into pRS202, resulting in pGO309. Materials and methods Microscopy Yeast strains and plasmid constructions GFP and FM 4-64 fluorescence and differential interference contrast (DIC) Standard protocols were used for culturing S. cerevisiae, cellular transfor- microscopy was performed using a DMRXA microscope (Leica) equipped mations, and spheroplast preparations (Guthrie and Fink, 2002). See Table with a Cooke Sensicam digital camera (Applied Scientific Instruments). Im- II for the genotypes of yeast strains used in this work. Gene deletions were ages were deconvolved using Slidebook software (Intelligent Imaging In- constructed by homologous recombination using site-specific deletion cas- novations) and processed using Adobe Photoshop 7.0 software (Adobe settes (Guthrie and Fink, 2002). Systems Inc.). Cells were stained with FM 4-64 using a pulse-chase proce- Table II. Yeast strains used in this work Strain Genotype Reference SEY6210 MAT leu2-3,112 ura3-52 his3∆ 200 trp1-∆ 901 lys2-∆ 801 suc2-∆ 9 Robinson et al., 1988 BHY10 SEY6210; leu2-3,112::pBHY11 Horazdovsky et al., 1994 ∆ CC GOY57 BHY10; bro1 This work KGY1 BHY10; bro1∆ ::HIS3 This work DMY1 SEY6210; BRO1-GFP::HIS3 This work DMY2 SEY6210; BRO1-GFP::HIS3 vps4∆ ::TRP1 This work ∆ CC DBY15 BHY10; bro1 -GFP::HIS3 This work ∆ CC DBY18 BHY10; bro1 -GFP::HIS3 vps4∆ ::TRP1 This work GOY23 SEY6210; pep4∆ ::LEU2 prb1∆ ::LEU2 This work ∆ CC GOY71 GOY23; bro1 This work DBY43 GOY23; bro1∆ ::HIS3 This work GOY74 SEY6210; DOA4-GFP::HIS3 This work GOY75 SEY6210; DOA4-GFP::HIS3 vps4∆ ::TRP1 This work ∆ CC GOY88 SEY6210; DOA4-GFP::HIS3 bro1 This work ∆ CC GOY93 SEY6210; DOA4-GFP::HIS3 bro1 vps4∆ ::TRP1 This work GOY82 SEY6210; DOA4-GFP::HIS3 bro1∆ ::KAN This work GOY83 SEY6210; DOA4-GFP::HIS3 bro1∆ ::KAN vps4∆ ::TRP1 This work GOY69 SEY6210; BRO1-GFP::HIS3 doa4∆ ::KAN This work GOY70 SEY6210; BRO1-GFP::HIS3 doa4∆ ::KAN vps4∆ ::TRP1 This work GOY113 SEY6210; DOA4-GFP::KAN vps4∆ ::TRP1 vps24∆ ::HIS3 This work GOY114 SEY6210; DOA4-GFP::KAN vps4∆ ::TRP1 snf7∆ ::HIS3 This work GOY107 GOY23; DOA4-GFP::KAN BRO1-HA::HIS3 This work GOY108 GOY23; DOA4-GFP::KAN BRO1-HA::HIS3 vps4∆ ::TRP1 This work ∆ CC GOY106 GOY23; DOA4-GFP::KAN bro1 -HA::HIS3 This work ∆ CC GOY105 GOY23; DOA4-GFP::KAN bro1 -HA::HIS3 vps4∆ ::TRP1 This work DBY42 BHY10; vps4∆ ::TRP1 This work NLY25 BHY10; bro1∆ ::HIS3 vps4∆ ::TRP1 This work ∆ CC DBY24 BHY10; bro1 vps4∆ ::TRP1 This work GOY86 BHY10; vps23∆ ::HIS3 This work NLY10 BHY10; vps28∆ ::HIS3 This work NLY8 BHY10; vps37∆ ::HIS3 This work GOY85 BHY10; vps22∆ ::HIS3 This work GOY78 BHY10; vps25∆ ::HIS3 This work CRY2 BHY10; vps36∆ ::HIS3 This work GOY79 BHY10; vps2∆ ::HIS3 This work CRY1 BHY10; vps24∆ ::HIS3 This work GOY80 BHY10; vps20∆ ::HIS3 This work GOY81 BHY10; snf7∆ ::HIS3 This work GOY87 BHY10; vps27∆ ::HIS3 This work GOY91 BHY10; nhx1∆ ::HIS3 This work GOY90 BHY10; vps60∆ ::HIS3 This work 728 The Journal of Cell Biology | Volume 166, Number 5, 2004 dure at 30C as described previously (Odorizzi et al., 2003). 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Published: Aug 30, 2004

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