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Mol Biol Cell. Feb 2007; 18(2): 646–657.
PMCID: PMC1783776

Mvb12 Is a Novel Member of ESCRT-I Involved in Cargo Selection by the Multivesicular Body PathwayAn external file that holds a picture, illustration, etc.
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Sandra Lemmon, Monitoring Editor

Abstract

The multivesicular body (MVB) sorting pathway impacts a variety of cellular functions in eukaryotic cells. Perhaps the best understood role for the MVB pathway is the degradation of transmembrane proteins within the lysosome. Regulation of cargo selection by this pathway is critically important for normal cell physiology, and recent advances in our understanding of this process have highlighted the endosomal sorting complexes required for transport (ESCRTs) as pivotal players in this reaction. To better understand the mechanisms of cargo selection during MVB sorting, we performed a genetic screen to identify novel factors required for cargo-specific selection by this pathway and identified the Mvb12 protein. Loss of Mvb12 function results in differential defects in the selection of MVB cargoes. A variety of analyses indicate that Mvb12 is a stable member of ESCRT-I, a heterologous complex involved in cargo selection by the MVB pathway. Phenotypes displayed upon loss of Mvb12 are distinct from those displayed by the previously described ESCRT-I subunits (vacuolar protein sorting 23, -28, and -37), suggesting a distinct function than these core subunits. These data support a model in which Mvb12 impacts the selection of MVB cargoes by modulating the cargo recognition capabilities of ESCRT-I.

INTRODUCTION

The endosomal system coordinates protein trafficking between various subcellular compartments, including the Golgi, plasma membrane, and lysosome. Cell surface proteins, including activated receptors, that have undergone endocytosis are typically recycled to the plasma membrane or targeted deeper into the endosomal pathway for degradation within the lysosome. Endosomal membrane proteins destined for the lumen of the lysosome undergo an additional sorting reaction during their inclusion into the multivesicular body (MVB) pathway (for review, see Gruenberg and Stenmark, 2004 blue right-pointing triangle; Babst, 2005 blue right-pointing triangle). MVBs form when the limiting membrane of the late endosome invaginates and buds into the lumen of the organelle, actively selecting a subset of the proteins from the limiting membrane in the process (Gorden et al., 1978 blue right-pointing triangle; Haigler et al., 1979 blue right-pointing triangle). The intralumenal vesicles within the MVB and their contents are exposed to hydrolases within the lysosome as a consequence of heterotypic fusion of these organelles. By contrast, proteins within the limiting membrane of the MVB are delivered to the limiting membrane of the lysosome/vacuole after heterotypic fusion (Odorizzi et al., 1998 blue right-pointing triangle).

Sorting of cargo into the MVB pathway and heterotypic fusion with the lysosme/vacuole leads to delivery into the hydrolytic lumen of the organelle, and therefore entry into this pathway must be highly regulated. Ubiquitination is the best-characterized cis-acting signal mediating entry into the MVB pathway (for reviews, see Katzmann et al., 2002 blue right-pointing triangle; Hicke and Dunn, 2003 blue right-pointing triangle; Raiborg et al., 2003 blue right-pointing triangle). Studies in organisms ranging from the yeast Saccharomyces cerevisiae, to mammalian cells have demonstrated that ubiquitin modification of a variety of MVB cargoes is a requisite for entry into this pathway, and ubiquitin modification of endosomal membrane proteins that are not normally MVB cargoes is sufficient to target them into this pathway (Katzmann et al., 2001 blue right-pointing triangle; Reggiori and Pelham, 2001 blue right-pointing triangle; Urbanowski and Piper, 2001 blue right-pointing triangle; Raiborg et al., 2002 blue right-pointing triangle). Ubiquitin-independent cargoes of the MVB pathway have also been described, but relevant signals for inclusion have not been precisely defined. In yeast, ubiquitin modification of the Sna3 protein has been reported to be dispensable for Sna3 entry into the MVB pathway (Reggiori and Pelham, 2001 blue right-pointing triangle). In the case of the mammalian melanosomal protein Pmel17, the relevant sorting information for targeting to intralumenal vesicles resides within the lumenal domain of the protein (Theos et al., 2006 blue right-pointing triangle), suggesting a novel mechanism by which this cargo is selected.

The sorting of all yeast MVB cargoes requires the function of the class E vacuolar protein sorting (Vps) proteins as the trans-acting machinery (Odorizzi et al., 1998 blue right-pointing triangle). This machinery has been conserved through evolution from yeast to humans (for reviews, see Katzmann et al., 2002 blue right-pointing triangle; Babst, 2005 blue right-pointing triangle) and has been demonstrated to mediate the budding of retroviruses such as human immunodeficiency virus-1 (Morita and Sundquist, 2004 blue right-pointing triangle). The class E Vps proteins recognize ubiquitinated MVB cargo proteins and actively sort them into the MVB pathway (for reviews, see Katzmann et al., 2002 blue right-pointing triangle; Raiborg et al., 2003 blue right-pointing triangle; Babst, 2005 blue right-pointing triangle). In class E vps mutants, MVBs fail to form and therefore MVB cargoes fail to reach the vacuolar lumen. (Raymond et al., 1992 blue right-pointing triangle; Odorizzi et al., 1998 blue right-pointing triangle). Many of the class E Vps proteins form the endosmal sorting complexes required for transport (ESCRTs). ESCRT-I, -II, and -III, together with additional class E Vps proteins such as Vps27/Hrs and Vps4/SKD1, transiently associate with the endosomal membrane to execute the MVB sorting reaction (Katzmann et al., 2001 blue right-pointing triangle; Babst et al., 2002a blue right-pointing triangle,b blue right-pointing triangle; Bache et al., 2003 blue right-pointing triangle; Bilodeau et al., 2003 blue right-pointing triangle; Katzmann et al., 2003 blue right-pointing triangle; von Schwedler et al., 2003 blue right-pointing triangle; Bowers et al., 2004 blue right-pointing triangle). The class E proteins Vps23/Tsg101, Vps28, and Vps37 were described previously as subunits of the 350-kDa ESCRT-I complex, which plays a role in the recognition of ubiquitin-modified MVB cargoes through the ubiquitin E2 variant (UEV) domain of Vps23/Tsg101 (Babst et al., 2000 blue right-pointing triangle; Bishop and Woodman, 2001 blue right-pointing triangle; Katzmann et al., 2001 blue right-pointing triangle; Bache et al., 2004 blue right-pointing triangle; Teo et al., 2004 blue right-pointing triangle). Recent structural determination of the ESCRT-I core suggests that Vps23, Vps28, and Vps37 may be present in equal molar ratios within this complex (Kostelansky et al., 2006 blue right-pointing triangle; Teo et al., 2006 blue right-pointing triangle); however, it is unclear how these subunits of 43, 28, and 25 kDa alone would comprise a complex with an apparent molecular mass of 350 kDa. ESCRT-I recruitment to the endosomal membrane is dependent upon its association with Vps27/Hrs, which seems to dictate site selection for the MVB sorting reaction by virtue of its ability to bind both the endosomally enriched lipid phosphatidylinositol-3-phosphate [PtdIns(3)P] as well as ubiquitin modified MVB cargo through ubiquitin interacting motifs (Bache et al., 2003 blue right-pointing triangle; Bilodeau et al., 2003 blue right-pointing triangle; Katzmann et al., 2003 blue right-pointing triangle).

Cellular components involved in mediating ubiquitin-dependent entry into the MVB pathway have begun to be elucidated. In yeast, deletion of class E vps genes results in a dramatic missorting of all MVB cargoes; however, mutants specifically defective for the delivery of ubiquitin-dependent MVB cargoes retain the ability to sort ubiquitin-independent MVB cargoes, including Sna3 (Bilodeau et al., 2002 blue right-pointing triangle; Katzmann et al., 2004 blue right-pointing triangle). This suggests additional mechanisms of cargo selection by this pathway exist. To gain insight into this process, we performed a genetic screen for loci affecting the MVB sorting of Sna3, a putative ubiquitin-independent MVB cargo (Reggiori and Pelham, 2001 blue right-pointing triangle; Katzmann et al., 2004 blue right-pointing triangle). This screen identified Mvb12 as a critical factor for sorting a subset of MVB cargoes, including Sna3 and Ste3. However, trafficking of other MVB cargoes such as carboxypepsidase S (CPS) and Ste2 is largely unaffected in mvb12Δ cells, as is general endosomal function. Several lines of evidence indicate that Mvb12 impacts MVB cargo sorting as a novel subunit of the previously described ESCRT-I. The distinct phenotypes of mvb12Δ compared with deletion of other ESCRT-I subunits suggest that Mvb12 is a modulator of the Vps23/28/37 ESCRT-I core machinery's ability to sort specific MVB cargoes. These results identify Mvb12 as a novel subunit of ESCRT-I that modulates the cargo sorting activity of this complex.

MATERIALS AND METHODS

S. cerevisiae strains used in this study are described in Table 1. The collection of viable deletion mutants was obtained from Research Genetics (Huntsville, AL). Methodologies and reagents described previously for the purpose of performing genome-wide synthetic genetic interaction analyses (Tong et al., 2001 blue right-pointing triangle) were used to introduce Sna3-GFP into the collection of viable deletion mutants (Tong et al., 2001 blue right-pointing triangle) Double deletion mutants in which both deletions are marked with HIS3 were generated by performing crosses between opposite mating types containing the relevant deletions, followed by sporulation, tetrad dissection, and verification of genotype.

Table 1.
Yeast strains used in this study

Plasmids

The MVB12-GFP fusion gene was polymerase chain reaction (PCR) amplified from genomic DNA from JPY29, cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA), and subcloned into the pRS415 and pRS414 vectors by using XbaI and SpeI sites. pRS416-Sna3-GFP has been described previously (Katzmann et al., 2004 blue right-pointing triangle). Sna3KallR-GFP was constructed through mutagenesis of pRS416-Sna3-GFP (GeneTailor; Invitrogen). GFP-CPS and Ste2-GFP have been described previously (Odorizzi et al., 1998 blue right-pointing triangle). Ste3-GFP and Ste3-GFP-Ub were graciously provided by Robert Piper (University of Iowa, Iowa City, IA). DsRed FYVE was described previously as an endosomal marker (Katzmann et al., 2003 blue right-pointing triangle). pMB103 (pRS416-Vps4E233Q) expresses a dominant-negative form of Vps4 that has been described previously (Babst et al., 1997 blue right-pointing triangle). The MVB12 open reading frame (ORF) was amplified from yeast genomic DNA and cloned using BamHI and XhoI sites into the pET28b (Novagen, Madison, WI) and pnTAP416 vectors digested with SalI and BamHI, for bacterial expression of His6-Mvb12 and yeast expression of TAP-Mvb12, respectively. To create pGPD414 HA-ubiquitin, pGDP416 HA-ubiquitin (Davies et al., 2003 blue right-pointing triangle) was digested with SacI and KpnI and subcloned into pRS414. The TAP tag was cloned into the EcoRI site of pGPD416 (Mumberg et al., 1995 blue right-pointing triangle) to create the pnTAP416 vector. All PCR-based plasmids were sequenced to ensure that no aberrant mutations were present.

Microscopy

Fluorescence microscopy was performed on live cells in minimal media, by using a Nikon fluorescence microscope with fluorescein isothiocyanate, rhodamine, green fluorescent protein (GFP), and DsRed filters and a digital camera (Coolsnap HQ; Photometrix, Melbourne, Australia). Images were deconvolved with Delta Vision software (Applied Precision, Seattle, WA). FM4-64 labeling was performed as described in Vida and Emr (1995) blue right-pointing triangle. Kinetic analysis of FM4-64 uptake was performed by labeling cells on ice for 15 min, followed by chasing at room temperature.

Biochemical Analyses

Gel filtration analyses were performed as described previously (Katzmann et al., 2001 blue right-pointing triangle). Bacterial expression of His6-Mvb12 was induced in the HMS174 DE3 strain at 37°C for 3.5 h with 0.5 mM isopropyl β-d-thiogalactoside. Crude lysate subjected to a 100,000 × g clearing spin was used for gel filtration. Tagged proteins were visualized using monoclonal antibodies anti-HA.11 (Covance, Princeton, NJ), monoclonal anti-GFP AV JL-8 (BD Biosciences, San Jose, CA), or penta-His (QIAGEN, Valencia, CA). Pulse-chase analysis of CPS, carboxypeptidase Y (CPY), Sna3-GFP, and Sna3KallR-GFP was performed as described previously (Babst et al., 2002a blue right-pointing triangle), and quantitation was performed using phosphorimaging screens and a Storm 840 system (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Protein A purification using TAP-Mvb12 was performed as described in Azmi et al. (2006) blue right-pointing triangle, with the exception that 15 OD600 of cells were lysed in phosphate-buffered saline. Proteins were visualized with polyclonal anti-Vps23 and anti-Vps28 (Katzmann et al., 2001 blue right-pointing triangle). Ste3 immunoprecipitation was performed essentially as described in Katzmann et al. (2001) blue right-pointing triangle, with all buffers containing 5 mM N-ethylmaleimide and incubations performed at 50°C. Yeast expressing Ste3-GFP and hemagglutinin (HA)-ubiquitin were trichloroacetic acid precipitated, processed, and lysed in urea cracking buffer with glass beads. Immunoprecipitation was performed with monoclonal anti-GFP AV JL-8 (BD Biosciences), and samples were subjected to SDS-PAGE and Western blotting. Ste3 was detected with anti-GFP, and the ubiquitination status was determined with monoclonal anti-HA.11 (Covance) to recognize HA-ubiquitin.

RESULTS

Identification of Mvb12 as an MVB Sorting Factor

Function of the MVB pathway requires the activity of the class E Vps proteins, many of which assemble into the oligomeric ESCRTs. The large number of class E vps mutants that yield the same phenotype has made standard genetic approaches to identify new components impractical due to the reisolation of previously known factors. However, screening of the commercially available collection of viable yeast deletion mutants represents a rational method by which to identify additional nonessential genes required for the function of the MVB pathway. Sna3 has been described as a cargo that does not require ubiquitination to be sorted into the MVB pathway (Reggiori and Pelham, 2001 blue right-pointing triangle); thus, trafficking of Sna3 fused to GFP (Sna3-GFP) was analyzed to identify novel cargo recognition factors and components of the MVB pathway. Methodologies described previously to perform genome-wide synthetic genetic interaction studies (Tong et al., 2001 blue right-pointing triangle) were used to introduce Sna3-GFP into the collection of viable yeast deletion strains and fluorescence microscopy was used to analyze subcellular localization of Sna3-GFP in 4570 mutants. Whereas the total number of mutants within the collection is slightly higher than the number analyzed, mutants that failed to come through the selection procedure and were previously known to function in protein trafficking or seemed irrelevant were not pursued. For this reason, as well as essential genes not being included in this collection and the collection itself containing errors, this analysis is not intended to represent an exhaustive survey of all gene products required for the function of the MVB pathway. The majority of strains within the collection displayed wild-type Sna3-GFP distribution, indistinguishable from wild type wherein fluorescence is localized to the lumen of the vacuole (Figure 1; data not shown). However, a large number of previously known vps mutants were identified, validating the experimental procedure (Supplemental Figure 1). In addition to previously known trafficking factors, this approach identified two mutants that displayed MVB sorting defects distinct from previously characterized class E vps mutants: vta1Δ (Azmi et al., 2006 blue right-pointing triangle) and deletion of YGR206w (Figure 1 and Supplemental Figure 1). YGR206w encodes a 12-kDa protein that plays a role in MVB sorting and is referred to as MVB12.

Figure 1.
MVB12 is required for sorting of a subset of MVB cargoes. Genetic screening identified mvb12Δ as defective for the sorting of Sna3-GFP in the BY4742 genetic background. GFP-tagged forms of the MVB cargoes Sna3, Sna3KallR, CPS, and Ste3 were expressed ...

To allow direct comparison with previously characterized class E vps mutants, and to validate the identification of mvb12Δ by using the deletion collection, the MVB12 gene was deleted in our standard genetic background (SEY6210) and MVB sorting phenotypes were examined in greater detail. Cells were also stained with the fluorescent vacuolar dye FM4-64 to enable visualization of the limiting membrane of the vacuole. As in the BY4742 genetic background, analysis of Sna3-GFP distribution revealed an intermediate MVB sorting phenotype, with a portion mislocalized to perivacuolar structures (indicated by arrows in Figure 2). This Sna3-GFP localization pattern is distinct from both wild-type cells, wherein Sna3-GFP is largely within the vacuole lumen, or a class E vps mutant (vps23Δ), in which Sna3-GFP localized predominantly to the aberrant class E compartment (Figure 2). Moreover, the colocalization of Sna3-GFP and FM4-64 apparent in vps23Δ cells was absent in the mvb12Δ and wild-type cells. This suggests that the compartments to which Sna3-GFP localized in an mvb12Δ mutant are distinct from those seen in a standard class E vps mutant. This intermediate phenotype is consistent with the partial Sna3-GFP sorting defects observed in the mvb12Δ strain in the BY4742 genetic background, although less severe, and defects in both strains could be corrected by transforming a MVB12 plasmid (Figure 3; data not shown).

Figure 2.
Defects in sorting of MVB cargoes observed upon loss of MVB12 are distinct from loss of the ESCRT-I subunit VPS23. Isogenic wild-type (SEY6210), mvb12Δ and vps23Δ strains were used to analyze the MVB sorting of GFP-tagged MVB cargoes Sna3, ...
Figure 3.
Kinetic analysis of vacuolar protein delivery rates using pulse-chase immunoprecipitation. (A) Wild-type, mvb12Δ, or mvb12Δ cells expressing Mvb12-GFP that were also expressing Sna3-GFP or Sna3KallR-GFP were pulse labeled with [35S]Cys/Met, ...

To more quantitatively address the intermediate phenotype observed in mvb12Δ cells, kinetic analysis of Sna3-GFP sorting was performed by pulse-chase analysis (Figure 3A and Supplemental Figure 2A). Sna3-GFP is degraded within the vacuole lumen subsequent to sorting into the MVB pathway; hence, its turnover serves as a measure of its MVB sorting (Oestreich et al., 2007 blue right-pointing triangle). Consistent with microscopic analysis, loss of Mvb12 resulted in a decreased rate of turnover of Sna3-GFP (Figure 3A and Supplemental Figure 2A). Importantly, the presence of Sna3-GFP in the vacuolar lumen in the mvb12Δ cells, as well as the eventual maturation of Sna3-GFP, indicated that Mvb12 is not absolutely required for function of the MVB pathway but that Mvb12 does play a role in Sna3 MVB sorting. This is distinct from the class E vps mutant vps23Δ wherein Sna3-GFP is not observed within the vacuolar lumen and its turnover is stabilized (Figure 2 and Supplemental Figure 2A).

The role of Mvb12 in MVB sorting was explored by analyzing the trafficking of additional MVB cargoes in the mvb12Δ strain. CPS is a well-characterized MVB cargo in yeast (Odorizzi et al., 1998 blue right-pointing triangle). Ubiquitin modification of CPS by the ubiquitin ligase Rsp5 confers ubiquitin-dependent MVB sorting of CPS in a manner dependent upon ESCRTs (Katzmann et al., 2001 blue right-pointing triangle, 2004 blue right-pointing triangle); hence, CPS sorting serves as an indicator of ubiquitin-dependent MVB sorting. Localization of a GFP-CPS reporter was examined in wild type, mvb12Δ, and the class E mutant vps23Δ strains. Whereas the mvb12Δ strain exhibited pronounced Sna3-GFP localization to punctate structures, GFP-CPS localization was only subtly perturbed, displaying minor missorting to the limiting membrane of the vacuole and no obvious class E compartment in both genetic backgrounds (Figures 1 and and2).2). This can be readily observed in the merged images, wherein the vacuolar limiting membrane looks yellow in both mvb12Δ and vps23Δ cells (Figure 2). Kinetic analysis of CPS maturation in the mvb12Δ strain (JPY22) yielded a consistent result with only a modest delay evident (Figure 3B). Maturation of the soluble vacuolar protease CPY is also only partially impeded in the mvb12Δ strain (Figure 3C). Furthermore, the mvb12Δ strain displayed very low levels of secreted p2CPY similar to wild-type cells, in contrast to the large portion secreted in the class E vps mutant vps23Δ (Figure 3D). The kinetic delay of CPY processing observed for mvb12Δ cells in Figure 3C can also be observed in Figure 3D wherein p2CPY is present in the intracellular fraction at 20 min, but it has been converted to mature (and not secreted) by 40 min. In contrast, the vps23Δ strain exhibited extensive limiting membrane and class E compartment localization for both GFP-CPS and Sna3-GFP (Figure 2) and defects both in CPS and CPY maturation have been observed previously (Figure 3 and Supplemental Figure 2; Babst et al., 2000 blue right-pointing triangle). As has been observed previously, the class E vps mutants processed CPS to an aberrant form that migrates distinctly from wild-type mature (m)CPS. However, mvb12Δ cells did not display this misprocessed form; instead, mvb12Δ cells displayed mCPS indistinguishable from wild-type cells (Babst et al., 2002a blue right-pointing triangle; Supplemental Figure 2D). These results indicate that mvb12Δ displays distinct phenotypes from previously characterized class E vps mutants.

To further explore these distinctions, endocytic trafficking of the G protein-coupled receptors Ste2 and Ste3 was examined in the mvb12Δ strains of the appropriate mating type. Ubiquitin modification of Ste2 has been demonstrated to play a critical role in its internalization from the cell surface and subsequent delivery into the MVB pathway, resulting in delivery to the vacuolar lumen (Terrell et al., 1998 blue right-pointing triangle). Ste3, by contrast, seems to undergo recycling between the plasma membrane and an endosomal compartment until ubiquitination removes it from the recycling loop by targeting it into the MVB pathway (Chen and Davis, 2002 blue right-pointing triangle). The intracellular trafficking itineraries of Ste2 and Ste3 seem to be distinct; thus, both cargoes were analyzed in the mvb12Δ background. Analysis of Ste2-GFP localization in the mvb12Δ strain indicated that Mvb12 does not play a crucial role in this process, because no difference was observed compared with wild-type cells (Figure 4A). By contrast, trafficking of Ste3-GFP to the vacuolar lumen is severely impaired in the mvb12Δ strain, displaying vacuolar limiting membrane and perivacuolar punctate structure localization (Figure 4, A and C). Again, whereas mvb12Δ cells display Ste3-GFP localization to perivacuolar compartments, these cells do not colocalize with FM4-64 to the same degree observed in the class E vps23Δ cells. However, fusion of ubiquitin to Ste3-GFP (Ste3-GFP-Ub) can bypass the requirement for Mvb12 in Ste3 trafficking (Figure 4A). These results suggest that loss of Mvb12 negatively impacts the MVB sorting of Sna3 and Ste3 to a greater degree than sorting of CPS, Ste2, or Ste3-Ub. One explanation for the differential defects on Ste3-GFP and Ste3-GFP-Ub in mvb12Δ cells could be a defect in ubiquitin modification of Ste3. This was directly analyzed by immunoprecipitation of Ste3-GFP from wild-type, mvb12Δ, and vps23Δ cells expressing HA-ubiquitin, followed by Western blotting with either anti-GFP or anti-HA (Figure 4B). Although equivalent numbers of cells were used in for each immunoprecipitation, Western blotting with anti-GFP consistently revealed higher levels of Ste3-GFP in both mvb12Δ cells and vps23Δ cells compared with wild-type cells, consistent with a defect in its degradation in these backgrounds (Figure 4B). Regardless, mvb12Δ cells display an increased level of ubiquitinated Ste3-GFP compared with both wild-type and vps23Δ cells (Figure 4B), indicating that mvb12Δ cells are not defective for the ubiquitination of Ste3. Whereas the mechanism by which Mvb12 functions is not clear from these studies, these results indicate that that some MVB cargoes are more sensitive to Mvb12 function than others. This characteristic is distinct from prototypical class E vps mutants and suggests that Mvb12 may be functioning as an accessory factor involved in entry of a subset of cargoes into the MVB pathway.

Figure 4.
Loss of Mvb12 displays differential phenotypes on endocytic cargoes. (A) Ste3-GFP displayed a severe missorting phenotype in the mvb12Δ background, whereas Ste2-GFP seemed indistinguishable from wild-type cells. A chimera wherein ubiquitin is ...

To gain additional insight into the role of Mvb12 in MVB sorting, trafficking of Sna3 in the mvb12Δ strain was examined further. Sna3 sorting has been suggested to occur by a ubiquitin-independent process (Reggiori and Pelham, 2001 blue right-pointing triangle). However, more recent observations indicate that Sna3 is ubiquitylated (Peng et al., 2003 blue right-pointing triangle) and that both ubiquitin-dependent and ubiquitin-independent pathways can mediate Sna3 trafficking (Oestreich et al., 2007 blue right-pointing triangle). Analysis of CPS, Ste2-GFP, and Ste3-GFP-Ub trafficking indicated that Mvb12 does not play an essential role in the general entry of ubiquitin-dependent MVB cargoes into the MVB pathway. Thus, we hypothesized that Mvb12 may contribute more to the sorting process that does not require Sna3 ubiquitination. To examine this possibility, trafficking of Sna3-GFP with all lysines mutated to arginine (Sna3KallR-GFP) was examined in the mvb12Δ strain. Whereas Sna3KallR-GFP sorted in a manner indistinguishable from Sna3-GFP in the wild-type strain (SEY6210 and BY4742), mutation of the lysine residues resulted in enhanced Sna3KallR-GFP localization to peri-vacuolar punctate structures upon deletion of MVB12 compared with Sna3-GFP (Figures 1 and and2)2) and a delay in Sna3KallR-GFP cleavage (Figure 3, A and B, and Supplemental Figure 2, A and B). These results are consistent with Sna3 trafficking into the MVB via alternative pathways involving either ubiquitination of Sna3 or a process that does not require ubiquitination of Sna3 (Oestreich et al., 2007 blue right-pointing triangle), with loss of Mvb12 having a greater impact on the latter. The explanation for this observation is not clear at present, but it is interesting to note that Ste3 may also use this pathway. Alternatively, both Mvb12 and Sna3-ubiquitination may affect the kinetics of Sna3 entry into the MVB pathway and thus yield a synthetic phenotype when both are compromised. Although further dissection of Sna3 trafficking and Mvb12 function will be required to resolve these models, these observations are consistent with Mvb12 functioning to facilitate the sorting of a subset of MVB cargoes.

Endosomal Localization of Mvb12 Is Dependent upon the ESCRT-I Subunit Vp s23

Loss of Mvb12 function conferred several phenotypes associated with endosome-to-vacuole protein sorting defects, including partial defects in MVB sorting. Subcellular localization was addressed by integrating the GFP coding sequence in-frame with the chromosomal copy of MVB12, resulting in a functional Mvb12-GFP chimera (Figure 3, A–C). Visualization of cells expressing Mvb12-GFP revealed both cytoplasmic and intracellular punctate structures that colocalize with the endosomal marker DsRed-FYVE but not the Golgi marker Sec7-RFP (Figure 5). To further characterize these compartments, kinetic analysis of FM4-64 uptake was analyzed in cells expressing Mvb12-GFP (Figure 6). Colocalization of Mvb12-GFP with FM4-64 structures at early time points (1–3 min) was minimal, but increased from 5 to 7 min (Figure 6). At later time points (15–25 min), the discreet patterns of FM4-64 and Mvb12-GFP indicate that the Mvb12-GFP endocytic structures are largely perivacuolar. Colocalization with endosomes places Mvb12-GFP at a location where the MVB sorting reaction is occurring, consistent with the observed phenotypes upon its deletion. This localization is also consistent with results obtained in a previous proteome-wide localization analysis (Huh et al., 2003 blue right-pointing triangle).

Figure 5.
Mvb12 localizes to PtdIns(3)P-positive endosomes. Cells expressing Mvb12-GFP from their chromosomal locus were transformed with markers for either the Golgi (Sec7-RFP) or endosomes (DsRed-FYVE) and visualized by fluorescence microscopy. Bar, 5 μm. ...
Figure 6.
Mvb12 localizes with endosomes marked by short time course labeling with FM4-64. Wild-type cells expressing Mvb12-GFP were labeled with FM4-64 on ice, chased for the indicated time, and visualized by fluorescence microscopy. Bar, 5 μm.

Because MVB sorting defects were observed upon deletion of MVB12, an interaction with ESCRT machinery was explored by examining the subcellular localization of Mvb12-GFP in a variety of strains deleted for class E VPS genes (Figure 7 and Supplemental Figure 3). This analysis was performed by either crossing the chromosomally integrated MVB12-GFP reporter into various class E vps mutants or by transforming a plasmid-borne MVB12-GFP under the control of its endogenous promoter into the mutants; consistent results were obtained by either method. In bro1/vps31Δ, hse1Δ and the ESCRT-III-like mutants (fti1/vps46/did2Δ and vps60/mos10Δ), Mvb12-GFP distribution seemed indistinguishable from wild-type cells (Supplemental Figure 3A). This suggests that these factors play no role in the endosomal association of Mvb12. However, loss of ESCRT-II (vps25Δ), ESCRT-III (vps20Δ, vps24Δ, or snf7Δ) or the AAA-ATPase Vps4 (vps4Δ) resulted in enhanced endosomal localization of Mvb12-GFP (Figure 7A and Supplemental Figure 3B). These results indicate that these factors are not required for the recruitment of Mvb12 to the endosomal membrane and are consistent with the idea that Mvb12 removal from the endosomal membrane requires the function of ESCRT-II, -III, and Vps4. In striking contrast, loss of Vps27 or the ESCRT-I subunits Vps23 and Vps37 resulted in a dramatic reduction in the amount of endosome-associated Mvb12-GFP (Figure 7A and Supplemental Figure 3A). To further address the redistribution of Mvb12 upon loss of the ESCRT-I subunit Vps23, a previously characterized dominant-negative form of Vps4 (Vps4E233Q) (Babst et al., 1997 blue right-pointing triangle) was expressed in the vps23Δ strain. Expression of Vps4E233Q in SEY6210 resulted in the enhanced accumulation of Mvb12-GFP on endosomes as well as reduced cytoplasmic distribution, consistent with Mvb12 localization in the vps4Δ strain; however, this accumulation was blocked in the vps23Δ strain, suggesting that even transient Mvb12 membrane association is absent without Vps23 (Figure 7B). However, loss of the ESCRT-I subunit Vps28 conferred increased membrane association of Mvb12-GFP, as observed upon loss of ESCRT-II, -III, and Vps4 (Figure 7A and Supplemental Figure 3B). This effect phenocopies the redistribution of Vps23-GFP that has been observed in the vps28Δ strain (Li et al., 1999 blue right-pointing triangle). These observations are consistent with Mvb12 membrane association requiring the function of Vps27 and the ESCRT-I subunits Vps23 and Vps37. ESCRT-I membrane association is dependent upon Vps27, but Vps27 membrane association is independent of ESCRT-I (Katzmann et al., 2003 blue right-pointing triangle). Similarly, association of GFP-Vps27 with endosomes marked by DsRed-FYVE is not disrupted in mvb12Δ cells (Supplemental Figure 3C). Thus, Mvb12 behaves in a manner similar to the ESCRT-I subunit Vps23. These observations raised the possibility that Mvb12 is a novel subunit of ESCRT-I that facilitates its recognition of a subset of MVB cargoes.

Figure 7.
Mvb12-GFP localization is dependent upon ESCRT-I. (A) Mvb12-GFP localization mimics Vps23 localization. Wild-type, vps27Δ, vps23Δ, and vps4Δ cells expressing chromosomally integrated MVB12-GFP were visualized using fluorescence ...

Mvb12 Is a Component of ESCRT-I

To examine an association of Mvb12 with ESCRT-I, yeast extracts from cells expressing Mvb12-GFP or Mvb12-HA were first analyzed by gel filtration. Regardless of the form of the epitope tag used, Mvb12 behaved as a species of approximately 350 kDa, as has been demonstrated previously for ESCRT-I (Figure 8A; Babst et al., 2000 blue right-pointing triangle; Katzmann et al., 2001 blue right-pointing triangle). Monomeric Mvb12 would be predicted to behave as a species smaller than 45 kDa with either epitope and was not detected in yeast extracts (Figure 8A; data not shown), suggesting that it is stably associated with this higher molecular mass complex in vivo. Furthermore, bacterially expressed His6-Mvb12 behaves as a species of approximately 18 kDa when subjected to gel filtration (Figure 8A). Given the separation range of the Sephacryl S-300 gel filtration column, this observation is consistent with bacterially expressed Mvb12 behaving as a monomer. These results suggest that Mvb12 is stably associated with a higher molecular mass complex in yeast extracts.

Figure 8.
Mvb12 physically associates with ESCRT-I in a stable manner. (A) Sephacryl S-300 gel filtration analyses were performed on extracts prepared from a variety of yeast expressing Mvb12-GFP or bacterial extracts from cells expressing His6-Mvb12. Fractions ...

The apparent molecular mass of Mvb12-GFP in a variety of class E vps deletion backgrounds was analyzed by gel filtration to address their impact on the formation of the higher molecular mass complex. The apparent molecular mass of the Mvb12-GFP containing complex was unaffected in lysates generated from vps27Δ, vps25Δ (ESCRT-II), snf7Δ or vps24Δ (ESCRT-III), or vps4Δ strains (Figure 8A). However, elimination of the ESCRT-I subunits (vps23Δ, vps28Δ, and vps37Δ) reduced the apparent molecular mass to ~200 kDa in Vps23 or Vps28 and dramatically destabilized the Mvb12-GFP protein to the point that it was undetectable with loss of Vps37 (Figure 8A; data not shown). This result indicated that the formation of the Mvb12-containing complex depends on the function of the ESCRT-I complex. Because ESCRT-I is a 350-kDa complex, the simplest interpretation is that Mvb12 associates with ESCRT-I itself. Moreover, gel filtration analysis of Vps23 and Vps28 in extracts generated from the mvb12Δ strain revealed a reduction in the apparent molecular mass of ESCRT-I to less than 200 kDa (Figure 8B). For comparison, loss of Vps28 shifts the apparent molecular mass of Vps23 to approximately 200 kDa, whereas loss of Vps37 has an even greater effect on its apparent mass (Figure 8B). These observations support the conclusion that Mvb12 is a stable subunit of ESCRT-I.

To directly address the association of Mvb12 with ESCRT-I, isolation of a tandem affinity purification (TAP)-tagged form of Mvb12 was performed. TAP-Mvb12 was expressed in wild-type, vps23Δ, and vps37Δ strains, purified from cleared lysates under native conditions using IgG-Sepharose, and the isolated material was subjected to Western analysis with anti-Vps23 and anti-Vps28 antisera (Figure 8C). The ESCRT-I components Vps23 and Vps28 copurified with TAP-Mvb12 from the wild-type lysate (lane 6), but they did not copurify with the TAP-tag alone (lane 5). This result indicated that Mvb12 is physically associated with ESCRT-I. Elimination of Vps23 did not reduce TAP-Mvb12 isolation of Vps28 (lane 7) nor did loss of Vps28 eliminate TAP-Mvb12 isolation of Vps23 (Supplemental Figure 4). However, isolation of the remaining ESCRT-I components with TAP-Mvb12 was compromised in the vps37Δ strain (Figure 8C and Supplemental Figure 4). Loss of another component of the MVB sorting machinery (vps4Δ) did not affect the association between Mvb12 and ESCRT-I (Supplemental Figure 4), consistent with the gel filtration analysis of the Mvb12-containing complex in vps4Δ lysates. These results confirmed that Mvb12 is a novel component of ESCRT-I and are consistent with the isolation of Vps23 and Vps37 with Mvb12-TAP in the yeast proteome-wide analysis (Krogan et al., 2006 blue right-pointing triangle). However, the phenotypes of mvb12Δ differ from the phenotypes of vps23Δ, vps28Δ and vps37Δ. These results lead us to propose that Mvb12 is modulating the ability of ESCRT-I to mediate MVB sorting of specific cargoes rather than being a core component of ESCRT-I.

DISCUSSION

The degradative capacity of the MVB pathway dictates that cargo selection must be strictly regulated. Our understanding of this process has been expanded through the identification of ubiquitylation as a posttranslational modification that can target proteins into this pathway, as well as trans-acting machinery capable of recognizing this modification and directing entry in this pathway (for reviews, see Katzmann et al., 2002 blue right-pointing triangle; Hicke and Dunn, 2003 blue right-pointing triangle; Raiborg et al., 2003 blue right-pointing triangle; Babst, 2005 blue right-pointing triangle). ESCRT-I was first identified as a complex that is capable of interacting with ubiquitin-modified MVB cargo via the UEV domain in Vps23 (Katzmann et al., 2001 blue right-pointing triangle). Subsequent work has revealed the presence of additional members of the class E Vps proteins that contain ubiquitin-interacting domains, including Vps27/Hrs, which participates in recruitment of ESCRT-I (Bilodeau et al., 2002 blue right-pointing triangle; Shih et al., 2002 blue right-pointing triangle; Bache et al., 2003 blue right-pointing triangle; Bilodeau et al., 2003 blue right-pointing triangle; Katzmann et al., 2003 blue right-pointing triangle). Together, Vps27 and ESCRT-I seem to play a critical role in the selection of ubiquitin-modified MVB cargoes. The reason behind this apparently complicated mechanism of cargo recognition is not entirely obvious; however, a variety of explanations including site of action, degree/linkage of cargo ubiquitination, and even affinity have merits. Furthermore, cargoes that are capable of entering the MVB pathway independent of ubiquitin modification exist and may require additional mechanisms of recognition by the MVB sorting machinery.

To better understand the mechanisms of cargo selection by the MVB pathway we performed a genetic screen for factors impacting Sna3 sorting, identifying mvb12Δ in the process. Deletion of MVB12 confers cargo-specific defects in MVB sorting, a feature that makes it distinct from previously described class E vps deletion mutants that display unilateral defects in MVB cargo sorting. This feature is particularly interesting considering that our data indicate that Mvb12 is a member of the ESCRT-I complex. But given that loss of the core ESCRT-I subunits (Vps23, Vps28, and Vps37) confer significantly more severe phenotypes, Mvb12 is not critical for all functions executed by ESCRT-I. Based on these observations, we suggest that Mvb12 plays a role in assisting ESCRT-I during sorting of a subset of MVB cargoes.

Although further investigation is required to distinguish the mechanism by which this occurs, several possibilities exist that could explain this role in cargo-specific function. One potential model is that Mvb12 facilitates the binding of ESCRT-I to a subset of MVB cargoes, including Ste3 and Sna3, but it is dispensable for binding of ESCRT-I to CPS and Ste2. Although it is attractive to speculate that Mvb12 could directly bind to Ste3 and Sna3 and mediate their interaction with ESCRT-I, it is equally likely that Mvb12 is not involved in directly binding these cargoes but helps to generate the Ste3 and Sna3 binding site(s) within ESCRT-I indirectly. How would this model explain the suppression of Ste3 trafficking defects in the mvb12Δ strain by direct fusion to ubiquitin (Ste3-GFP-Ub)? Although Ste3 has been demonstrated to be a ubiquitin-dependent MVB cargo, there may be ways in which the ubiquitinated form of Ste3 is recognized by the Mvb12-containing ESCRT-I that are distinct from the ESCRT-I recognition of the Ste3-Ub fusion, possibly through differential recognition of ubiquitin itself. Further experimentation will be required to address the model that Mvb12 allows ESCRT-I binding, either directly or indirectly, to a subset of MVB cargoes.

A second model for the role of Mvb12 in the MVB sorting of Ste3 and Sna3 but not CPS or Ste2 involves the trafficking and MVB sorting of distinct cargoes via discrete endosomal compartments. Recent evidence in mammalian cells is consistent with the idea that there are distinct classes of MVBs being formed at distinct sites (White et al., 2006 blue right-pointing triangle). Similar phenomenon may be occurring in yeast. In this second model, Ste3 and Sna3 would traffic to the vacuole via an endosomal compartment that is distinct from that transited by CPS and Ste2. Although ESCRT-I mediates MVB sorting at both compartments, Mvb12 may be required for ESCRT-I localization or function at the Ste3/Sna3 compartment but not the Ste2/CPS compartment. Direct fusion of ubiquitin to the carboxy terminus of Ste3 (Ste3-Ub) may alter Ste3-Ub trafficking to the Ste2/CPS-sorting compartment, allowing MVB sorting in the absence of Mvb12. This idea seems plausible in that Ste2 is thought to internalize in a ubiquitinated form, whereas Ste3 is thought to recycle between an endosomal compartment and the cell surface until ubiquitination removes it from this recycling loop (Terrell et al., 1998 blue right-pointing triangle; Chen and Davis, 2002 blue right-pointing triangle). It is possible that endocytosed cargoes that are ubiquitinated bypass a recycling endosome. At present, characterization of the yeast endosomal system is not sufficient to address the possibility of discrete endosomes for sorting distinct MVB cargoes. Further experimentation will be required to map out the potential subtleties of the yeast endosomal system and cargo-specific MVB sorting.

A third model for the role of Mvb12 in cargo-specific sorting involves some more general form of modulation of ESCRT-I function, perhaps through associations with additional ESCRT machinery. For example, ESCRT-I efficiency could be impacted through Mvb12-modulated interactions with components that act upstream or downstream of ESCRT-I. How might this lead to cargo-specific defects upon loss of Mvb12? Different MVB cargoes, such as Sna3 and Ste3 may be more sensitive to the level of ESCRT-I function, thereby revealing differential sorting phenotypes when ESCRT-I function is compromised by loss of Mvb12. However, in our experience GFP-CPS is the most sensitive cargo that can be used to address function of the MVB pathway (Azmi et al., 2006 blue right-pointing triangle; our unpublished data). Thus, the observation that mvb12Δ has only a subtle impact on the sorting of GFP-CPS seems to cast doubt upon this third model. However, further experimentation will be required to definitively address this role for Mvb12 as a general modulator of ESCRT-I function.

Loss of Mvb12 function confers partial defects in the sorting of cargoes into the MVB pathway. Although the mechanism is unclear, Mvb12 seems to play a role in assisting ESCRT-I to sort a subset of MVB cargoes as a stable component of ESCRT-I. This begs the question as to how this was missed during the initial characterization of ESCRT-I. One possibility is that the small size of the Mvb12 protein (12 kDa) precluded its identification in the isolation procedure by using protein A-tagged Vps23 (Katzmann et al., 2001 blue right-pointing triangle). Although the reason that Mvb12 was not previously identified remains unclear, the more intriguing question that arises is whether mammalian ESCRT-I may also contain accessory subunits that have eluded detection to this point. Although Mvb12 has been conserved among fungi, sequence homologues in higher eukaryotes have escaped our detection. However, it seems likely that functional homologues exist that modulate the function of mammalian ESCRT-I with respect to specific MVB cargoes as well. Additional studies will provide a deeper understanding of the role of Mvb12 in cargo-specific ESCRT-I function during the MVB sorting reaction and may provide insight regarding mammalian counterparts as well.

Additional Note: The laboratories of both Markus Babst (University of Utah, Salt Lake City, UT) and Scott Emr (University of California, San Diego, San Diego, CA) have independently identified the gene product of YGR206w as a subunit of the ESCRT-I complex. Because it is a 12-kDa protein involved in MVB sorting, we have agreed on the designation Mvb12 for the gene product of YGR206w.

Supplementary Material

[Supplemental Material]

ACKNOWLEDGMENTS

We thank Chris Burd (University of Pennsylvania, Philadelphia, PA) for the giving us the courage to initiate a genome-wide screen to identify mutants incapable of properly sorting Sna3-GFP and critical discussions, Charlie Boone and Amy Tong (University of Toronto, Toronto, Ontario, Canada) for technical advice and reagents to execute screening of the deletion collection, and the Katzmann and Horazdovsky laboratories for helpful discussions. This work was supported by Grant R01 GM-73024-1 from the National Institutes of Health (to D.J.K.).

Abbreviations used:

CPS
carboxypeptidase S
CPY
carboxypeptidase Y
ESCRT
endosomal sorting complex required for transport
MVB
multivesicular body
ORF
open reading frame
PtdIns(3)P
phosphatidylinositol-3-phosphate
TAP
tandem affinity purification
Ub
ubiquitin
VPS
vacuolar protein sorting

Footnotes

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-07-0601) on December 6, 2006.

An external file that holds a picture, illustration, etc.
Object name is dbox.jpg The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

REFERENCES

  • Azmi I., Davies B., Dimaano C., Payne J., Eckert D., Babst M., Katzmann D. J. Recycling of ESCRTs by the AAA-ATPase Vps4 is regulated by a conserved VSL region in Vta1. J. Cell Biol. 2006;172:705–717. [PMC free article] [PubMed]
  • Babst M. A protein's final ESCRT. Traffic. 2005;6:2–9. [PubMed]
  • Babst M., Katzmann D. J., Estepa-Sabal E. J., Meerloo T., Emr S. D. Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Dev. Cell. 2002a;3:271–282. [PubMed]
  • Babst M., Katzmann D. J., Snyder W. B., Wendland B., Emr S. D. Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body. Dev. Cell. 2002b;3:283–289. [PubMed]
  • Babst M., Odorizzi G., Estepa E. J., Emr S. D. Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking. Traffic. 2000;1:248–258. [PubMed]
  • Babst M., Sato T. K., Banta L. M., Emr S. D. Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p. EMBO. J. 1997;16:1820–1831. [PMC free article] [PubMed]
  • Bache K. G., Brech A., Mehlum A., Stenmark H. Hrs regulates multivesicular body formation via ESCRT recruitment to endosomes. J. Cell Biol. 2003;162:435–442. [PMC free article] [PubMed]
  • Bache K. G., Slagsvold T., Cabezas A., Rosendal K. R., Raiborg C., Stenmark H. The growth-regulatory protein HCRP1/hVps37A is a subunit of mammalian ESCRT-I and mediates receptor down-regulation. Mol. Biol. Cell. 2004;15:4337–4346. [PMC free article] [PubMed]
  • Bilodeau P. S., Urbanowski J. L., Winistorfer S. C., Piper R. C. The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting. Nat. Cell Biol. 2002;4:534–539. [PubMed]
  • Bilodeau P. S., Winistorfer S. C., Kearney W. R., Robertson A. D., Piper R. C. Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome. J. Cell Biol. 2003;163:237–243. [PMC free article] [PubMed]
  • Bishop N., Woodman P. Tsg101/mammalian vps23 and mammalian vps28 interact directly and are recruited to vps4-induced endosomes. J. Biol. Chem. 2001;276:11735–11742. [PubMed]
  • Bonangelino C. J., Chavez E. M., Bonifacino J. S. Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae. Mol. Biol. Cell. 2002;13:2486–2501. [PMC free article] [PubMed]
  • Bowers K., Lottridge J., Helliwell S. B., Goldthwaite L. M., Luzio J. P., Stevens T. H. Protein-protein interactions of ESCRT complexes in the yeast Saccharomyces cerevisiae. Traffic. 2004;5:194–210. [PubMed]
  • Chen L., Davis N. G. Ubiquitin-independent entry into the yeast recycling pathway. Traffic. 2002;3:110–123. [PubMed]
  • Davies B. A., Topp J. D., Sfeir A. J., Katzmann D. J., Carney D. S., Tall G. G., Friedberg A. S., Deng L., Chen Z., Horazdovsky B. F. Vps9p CUE domain ubiquitin binding is required for efficient endocytic protein traffic. J. Biol. Chem. 2003;278:19826–19833. [PubMed]
  • Gorden P., Carpentier J. L., Cohen S., Orci L. Epidermal growth factor: morphological demonstration of binding, internalization, and lysosomal association in human fibroblasts. Proc. Natl. Acad. Sci. USA. 1978;75:5025–5029. [PMC free article] [PubMed]
  • Gruenberg J., Stenmark H. The biogenesis of multivesicular endosomes. Nat. Rev. Mol. Cell Biol. 2004;5:317–323. [PubMed]
  • Haigler H. T., McKanna J. A., Cohen S. Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J. Cell Biol. 1979;81:382–395. [PMC free article] [PubMed]
  • Hicke L., Dunn R. Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu. Rev. Cell Dev. Biol. 2003;19:141–172. [PubMed]
  • Huh W. K., Falvo J. V., Gerke L. C., Carroll A. S., Howson R. W., Weissman J. S., O'Shea E. K. Global analysis of protein localization in budding yeast. Nature. 2003;425:686–691. [PubMed]
  • Katzmann D. J., Babst M., Emr S. D. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell. 2001;106:145–155. [PubMed]
  • Katzmann D. J., Odorizzi G., Emr S. D. Receptor downregulation and multivesicular-body sorting. Nat. Rev. Mol. Cell Biol. 2002;3:893–905. [PubMed]
  • Katzmann D. J., Sarkar S., Chu T., Audhya A., Emr S. D. Multivesicular body sorting: ubiquitin ligase Rsp5 is required for the modification and sorting of carboxypeptidase S. Mol. Biol. Cell. 2004;15:468–480. [PMC free article] [PubMed]
  • Katzmann D. J., Stefan C. J., Babst M., Emr S. D. Vps27 recruits ESCRT machinery to endosomes during MVB sorting. J. Cell Biol. 2003;162:413–423. [PMC free article] [PubMed]
  • Kostelansky M. S., Sun J., Lee S., Kim J., Ghirlando R., Hierro A., Emr S. D., Hurley J. H. Structural and functional organization of the ESCRT-I trafficking complex. Cell. 2006;125:113–126. [PMC free article] [PubMed]
  • Krogan N. J., et al. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature. 2006;440:637–643. [PubMed]
  • Li Y., Kane T., Tipper C., Spatrick P., Jenness D. D. Yeast mutants affecting possible quality control of plasma membrane proteins. Mol. Cell. Biol. 1999;19:3588–3599. [PMC free article] [PubMed]
  • Morita E., Sundquist W. I. Retrovirus budding. Annu. Rev. Cell Dev. Biol. 2004;20:395–425. [PubMed]
  • Mumberg D., Muller R., Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 1995;156:119–122. [PubMed]
  • Odorizzi G., Babst M., Emr S. D. Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body. Cell. 1998;95:847–858. [PubMed]
  • Oestreich A. J., Aboian M., Lee J., Azmi I., Payne J., Issaka R., Davies B. A., Katzmann D. J. Characterization of multiple multivesicular body sorting determinants within Sna3: a role for the ubiquitin ligase Rsp5. Mol. Biol. Cell. 2007;18:707–720. [PMC free article] [PubMed]
  • Peng J., Schwartz D., Elias J. E., Thoreen C. C., Cheng D., Marsischky G., Roelofs J., Finley D., Gygi S. P. A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol. 2003;21:921–926. [PubMed]
  • Raiborg C., Bache K. G., Gillooly D. J., Madshus I. H., Stang E., Stenmark H. Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes. Nat. Cell Biol. 2002;4:394–398. [PubMed]
  • Raiborg C., Rusten T. E., Stenmark H. Protein sorting into multivesicular endosomes. Curr. Opin. Cell Biol. 2003;15:446–455. [PubMed]
  • Raymond C. K., Howald-Stevenson I., Vater C. A., Stevens T. H. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol. Biol. Cell. 1992;3:1389–1402. [PMC free article] [PubMed]
  • Reggiori F., Pelham H. R. Sorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting. EMBO J. 2001;20:5176–5186. [PMC free article] [PubMed]
  • Shih S. C., Katzmann D. J., Schnell J. D., Sutanto M., Emr S. D., Hicke L. Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis. Nat. Cell Biol. 2002;4:389–393. [PubMed]
  • Teo H., Gill D. J., Sun J., Perisic O., Veprintsev D. B., Vallis Y., Emr S. D., Williams R. L. ESCRT-I core and ESCRT-II GLUE domain structures reveal role for GLUE in linking to ESCRT-I and membranes. Cell. 2006;125:99–111. [PubMed]
  • Teo H., Veprintsev D. B., Williams R. L. Structural insights into endosomal sorting complex required for transport (ESCRT-I) recognition of ubiquitinated proteins. J. Biol. Chem. 2004;279:28689–28696. [PubMed]
  • Terrell J., Shih S., Dunn R., Hicke L. A function for monoubiquitination in the internalization of a G protein-coupled receptor. Mol. Cell. 1998;1:193–202. [PubMed]
  • Theos A. C., Truschel S. T., Tenza D., Hurbain I., Harper D. C., Berson J. F., Thomas P. C., Raposo G., Marks M. S. A lumenal domain-dependent pathway for sorting to intralumenal vesicles of multivesicular endosomes involved in organelle morphogenesis. Dev. Cell. 2006;10:343–354. [PMC free article] [PubMed]
  • Tong A. H., et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science. 2001;294:2364–2368. [PubMed]
  • Urbanowski J. L., Piper R. C. Ubiquitin sorts proteins into the intralumenal degradative compartment of the late-endosome/vacuole. Traffic. 2001;2:622–630. [PubMed]
  • Vida T. A., Emr S. D. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 1995;128:779–792. [PMC free article] [PubMed]
  • von Schwedler U. K., et al. The protein network of HIV budding. Cell. 2003;114:701–713. [PubMed]
  • White I. J., Bailey L. M., Aghakhani M. R., Moss S. E., Futter C. E. EGF stimulates annexin 1-dependent inward vesiculation in a multivesicular endosome subpopulation. EMBO J. 2006;25:1–12. [PMC free article] [PubMed]

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