QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

Vol. 18, Issue 2, 697-706, February 2007

This Article Abstract Full Text (PDF) Supplemental Materials All Versions of this Article: E06-08-0663v1 18/2/697    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by McNatt, M. W. Articles by Odorizzi, G. Search for Related Content PubMed PubMed Citation Articles by McNatt, M. W. Articles by Odorizzi, G.

Direct Binding to Rsp5 Mediates Ubiquitin-independent Sorting of Sna3 via the Multivesicular Body Pathway

Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347

Submitted August 1, 2006; Revised November 29, 2006; Accepted November 30, 2006
Monitoring Editor: Jean Gruenberg

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The sorting of most integral membrane proteins into the lumenal vesicles of multivesicular bodies (MVBs) is dependent on the attachment of ubiquitin (Ub) to their cytosolic domains. However, Ub is not required for sorting of Sna3, an MVB vesicle cargo protein in yeast. We show that Sna3 circumvents Ub-mediated recognition by interacting directly with Rsp5, an E3 Ub ligase that catalyzes monoubiquitination of MVB vesicle cargoes. The PPAY motif in the C-terminal cytosolic domain of Sna3 binds the WW domains in Rsp5, and Sna3 is polyubiquitinated as a consequence of this association. However, Ub does not appear to be required for transport of Sna3 via the MVB pathway because its sorting occurs under conditions in which its ubiquitination is impaired. Consistent with Ub-independent function of the MVB pathway, we show by electron microscopy that the formation of MVB vesicles does not require Rsp5 E3 ligase activity. However, cells expressing a catalytically disabled form of Rsp5 have a greater frequency of smaller MVB vesicles compared with the relatively broad distribution of vesicles seen in MVBs of wild-type cells, suggesting that the formation of MVB vesicles is influenced by Rsp5-mediated ubiquitination.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Multivesicular bodies (MVBs) are late endosomes containing lumenal vesicles that are formed by invagination of the endosomal membrane. The lumenal MVB vesicles are delivered into the hydrolytic interior of the lysosome upon fusion of the limiting MVB membrane with the lysosomal membrane (van Deurs et al., 1995; Futter et al., 1996; Mullock et al., 1998). Many cell-surface receptors that undergo down-regulation from the plasma membrane are sorted into MVB vesicles en route to being degraded in the lysosome, including members of the receptor tyrosine kinase family in metazoans and G protein–coupled receptors in the budding yeast Saccharomyces cerevisiae. In yeast, many biosynthetic proteins are also sorted into MVB vesicles during their transport from the Golgi to the vacuole, the functional equivalent of the lysosome (reviewed in Hicke and Dunn, 2003).

Most integral membrane proteins require the attachment of a single ubiquitin (Ub) to their cytosolic domains in order to be sorted into the MVB pathway (Katzmann et al., 2001; Reggiori and Pelham, 2001; Urbanowski and Piper, 2001). Sorting of monoubiquitinated MVB cargoes is mediated by a highly conserved machinery comprised of class E Vps proteins, many of which assemble into distinct endosomal sorting complexes required for transport (ESCRTs) that contain Ub-binding domains (reviewed in Hurley and Emr, 2006). In addition to Ub-mediated cargo recognition, class E Vps proteins are generally required for MVB vesicle budding because cells in which class E Vps proteins are disrupted contain malformed late endosomes that lack lumenal vesicles (Rieder et al., 1996; Odorizzi et al., 1998; Doyotte et al., 2005).

The class E Vps/ESCRT machinery is also required for the budding of many nonlytic enveloped viruses from infected host cells, which is topologically equivalent to the budding of MVB vesicles. Class E Vps proteins are recruited by virally encoded proteins that contain amino acid sequences known as "late domains" (reviewed in Morita and Sundquist, 2004). For example, the human immunodeficiency virus-1 Gag protein contains two late domains: the P(T/S)AP sequence, which binds the Tsg101 subunit of ESCRT-I, and the YPx_nL sequence, which binds Alix. Rous sarcoma virus and many other viruses have the PPxY late domain, which is a consensus binding site for WW domains (Macias et al., 2002). Viral PPxY late domains bind WW domains of the Nedd4 family of E3 Ub ligases, but the mechanistic role of Nedd4 proteins in viral budding is not clear (Morita and Sundquist, 2004).

Rsp5 is a Nedd4 homolog in yeast required for ubiquitination and sorting of most MVB cargo proteins (Blondel et al., 2004; Dunn et al., 2004; Hettema et al., 2004; Katzmann et al., 2004; Morvan et al., 2004). Sna3, however, is a biosynthetic cargo that does not require Rsp5 activity in order to be sorted into MVB vesicles (Katzmann et al., 2004), nor does it require Ub-binding functions of class E Vps proteins (Bilodeau et al., 2002). Nevertheless, MVB sorting of Sna3 is dependent on general functions of the class E Vps machinery (Reggiori and Pelham, 2001; Yeo et al., 2003; Katzmann et al., 2004). In mammalian cells, Ub-independent MVB cargo sorting can occur in a manner that is either dependent on class E Vps proteins (Tanowitz and Von Zastrow, 2002; Hislop et al., 2004) or independent of the class E Vps machinery (Theos et al., 2006). Thus, multiple mechanisms sort cargo proteins via the MVB pathway. Here, we show that MVB sorting of Sna3 is mediated by a PPxY sequence in its cytosolic domain that binds directly to the WW domains of Rsp5.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Yeast Strains and Plasmid Constructions
Standard protocols were used for manipulations of S. cerevisiae (Guthrie and Fink, 2002) and for DNA manipulations using Escherichia coli (Sambrook and Russell, 2001). All plasmids constructions were confirmed by DNA sequence analysis. See Table 1for the genotypes of yeast strains used in this study. Gene deletions and chromosomal integrations of epitope tags in yeast were constructed by homologous recombination of site-specific cassettes amplified by the PCR (Longtine et al., 1998). The doa4^C571S allele from pGO309 (Luhtala and Odorizzi, 2004) was subcloned into pRS306 (Sikorski and Hieter, 1989), resulting in plasmid pGO311, and then integrated into the genome of BYH10 (Horazdovsky et al., 1994) by homologous recombination (Guthrie and Fink, 2002) using pGO311 that had been linearized by restriction digestion, resulting in GOY100.

Preparation of Cell Extracts and Western Blotting
Yeast cell extracts were prepared from strains grown to OD₆₀₀ 0.5–0.8 and then harvested by centrifugation for 5 min at 500 x g, resuspended in 10 mM N-ethylmaleimide, and precipitated by the addition of 10% (vol/vol) trichloroacetic acid. After 10 min on ice, samples were isolated by centrifugation at 4°C for 10 min at 14,000 x g and then resuspended by sonication into ice-cold acetone. Samples were centrifuged and sonicated into ice-cold acetone again and centrifuged once more as described above, and then the pellets were dried by centrifugation under vacuum, resuspended by sonication into protein sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue), and 0.5 OD₆₀₀ units were resolved by SDS-PAGE.

For Western blotting, commercially available mouse monoclonal antibodies were used to detect the green fluorescence protein (GFP; Hoffman-La Roche, Nutley, NJ), the hemagglutinin epitope (HA; Zymed, South San Francisco, CA), and yeast 3-phosphoglycerate kinase (PGK; Invitrogen, Carlsbad, CA). Rabbit polyclonal antisera was used to detect yeast carboxypeptidase S (CPS; Cowles et al., 1997), and yeast Doa4. The anti-Doa4 antiserum was commercially prepared (Invitrogen) by coinjection of three peptide conjugates corresponding to amino acids 115-130, 620-635, and 894-909 of Doa4.

Fluorescence Microscopy
GFP, DsRed, and FM 4-64 fluorescence and differential interference contrast (DIC) microscopy was performed using a DMRXA microscope (Leica, Deerfield, IL) equipped with a Cooke Sensicam digital camera (Applied Scientific Instrumentation, Eugene, OR). Images were deconvolved using Slidebook 4.0 software (Intelligent Imaging Innovations, Denver, CO), and processed using Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA). Cells were stained with FM 4-64 (Invitrogen) using a pulse-chase procedure as described previously (Odorizzi et al., 2003).

Electron Microscopy
Cells were grown to logarithmic phase, then high-pressure frozen, freeze-substituted with 0.1% uranyl acetate, 0.25% glutaraldehyde, anhydrous acetone at –90°C, and embedded in Lowicryl HM20 (Polysciences, Warrington, PA) and subsequently polymerized under UV at –50°C (Winey et al., 1995). Thin sections (70 nm) were prepared by microtomy and examined on a Phillips CM10 transmission electron microscope (TEM; Mahwah, NJ) at 80 kV. For electron tomography, 200-nm semithick sections were placed on rhodium- plated Formvar-coated copper slot grids and mapped on the Phillips CM10 TEM at 80 kV, and then dual tilt series images were collected from +60° to –60° with 1° increments at 200 kV using a Tecnai 20 field emission gun (FEI, Beaverton, OR). Tomograms were imaged at 29,000x with a 0.77-nm pixel resolution (binning 2). Sections were coated on both sides with 15-nm fiducial gold for reconstruction of back projections using IMOD software (Kremer et al., 1996). 3dmod software was used for mapping structure surface areas. Mean z-scale values for wild-type and rsp5^G555D sections were within 3%. Best-fit sphere models were used to measure vesicle diameters to the outer leaflet of membrane bilayers. IMOD calculated limiting membrane surface areas using three-dimensional (3D) mesh structures derived from closed contours drawn each 3.85 nm using imodmesh.

Affinity Isolation of Recombinant Proteins in Vitro
Liquid cultures (500 ml) of the E. coli strain BL21(DE3) transformed with the Codon Plus IL plasmid (Stratagene, La Jolla, CA) and transformed with pET-His PL or pGEX-4T1 expression vectors were grown to logarithmic phase in a shaking water bath at 37°C, and then incubated for 20 min in a shaking water bath at 20°C. On addition of 0.5 mM isopropyl--D-thiogalactopyranoside, the cultures were incubated an additional 16–20 h at 20°C and then harvested by centrifugation at 4500 x g for 5 min at 4°C. Pelleted cells that had been transformed with pET-HIS PL expression vectors were resuspended in ice-cold phosphate-buffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.65) containing 1 mM phenylmethylsulfonylfluoride (PMSF), 10 mM 2-mercaptoethanol, 20 mM imidazole, and 1 mg/ml lysozyme, and then incubated on ice for 30 min before being subjected to sonication. The lysates were clarified of cell debris by centrifugation at 15,000 x g for 5 min at 4°C, followed by centrifugation at 25,000 x g for 10 min at 4°C. Talon Cobalt affinity resin (Clontech, San Jose, CA) was added to the resulting supernatant, which was then rotated for 1 h at 4°C, and then washed three times with binding buffer (lysis buffer without lysozyme or PMSF). The His₆ fusion proteins were subsequently eluted from the resin with PBS, pH 8.0 containing 250 mM imidazole.

Pelleted cells that had been transformed with pGEX-4T1 expression vectors were resuspended in ice-cold Tris-buffered saline (TBS; 0.5 M NaCl, 20 mM Tris-HCL, pH 7.5), 1.0% Triton X-100, 1 mM PMSF, and 1 mg/ml lysozyme and then incubated on ice for 30 min before being subjected to sonication. The lysates were clarified of cell debris as described above, and the resulting supernatants were filtered through a 0.45-µm pore size poly(vinylidene difluoride) membrane (Millipore, Bedford, MA) before passage through a GSTrap FF 5-ml column (GE Healthcare) equilibrated with TBS. After washing the column with 25 ml TBS, GST fusion proteins were eluted with 50 mM Tris-HCl, pH 8.0, containing 10 mM reduced glutathione.

Each isolated GST and His₆ fusion protein was assessed for its relative purity and concentration by SDS-PAGE and staining with Coomassie Brilliant Blue. Protein-binding assays were performed by adding pairwise combinations of the His₆ and GST fusion proteins (5 µg of each) to binding buffer (390 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4, 0.1% Triton X-100, 0.5% bovine serum albumin) to achieve a final volume of 500 µl, which was rotated at 4°C for 1 h. Afterward, 25 µl glutathione-Sepharose affinity resin (equilibrated in binding buffer) was added, and the mixture was rotated for 1 h at 4°C. The resin were then isolated by centrifugation at 10,000 x g for 2 min at 4°C, washed three times with ice-cold binding buffer, washed twice with ice-cold PBS, and dried by centrifugation under vacuum. The sample was then resuspended in protein sample buffer, resolved by SDS-PAGE, and observed by staining with Coomassie Brilliant Blue.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Rsp5 Mediates Polyubiquitination of Sna3
Ub-independent sorting of Sna3 via the MVB pathway was originally demonstrated by fluorescence microscopic localization of GFP fused to a mutant form of Sna3 in which its only two cytosolic lysine residues (K₁₉ and K₁₂₅; Figure 1A) had been substituted with arginine (Reggiori and Pelham, 2001). However, Sna3 was recently identified in two independent yeast proteomic studies as a substrate for polyubiquitination (Hitchcock et al., 2003; Peng et al., 2003). Therefore, we investigated whether Sna3-GFP is modified by Ub attachment en route to the vacuole. Western blot analysis using anti-GFP antiserum detected both the full-length fusion protein (48 kDa) and free GFP (25 kDa) in extracts of wild-type cells expressing Sna3-GFP (Figure 1B). GFP cleavage from Sna3 was blocked when activation of vacuolar proteases was prevented by deletion of the PEP4 gene (pep4), indicating that cleavage of Sna3-GFP occurred upon its delivery into the vacuole lumen (Figure 1B). Higher molecular-weight forms of full-length Sna3-GFP were also detected in extracts of pep4 cells (Figure 1B, asterisks). Consistent with these forms being due to polyubiquitination, their abundance was significantly increased upon replacement of the wild-type DOA4 gene with the catalytically inactive doa4^C571S allele (Figure 1B), indicating that the majority of Sna3-GFP is normally deubiquitinated by Doa4. Indeed, overexpression of wild-type DOA4 eliminated the higher molecular-weight forms of Sna3-GFP observed in extracts of pep4 cells (Figure 1C). These forms corresponded to polyubiquitination of Sna3 rather than GFP because a similar pattern of modification was observed in extracts of pep4 doa4^C571S cells expressing Sna3 fused to HA, and overexpression of Myc-tagged Ub, which is slightly larger than wild-type Ub, caused each of the higher molecular-weight forms of Sna3-HA to appear as a doublet (Figure 1D).

View larger version (30K): [in this window] [in a new window]   Figure 1. Sna3 is polyubiquitinated. (A) Schematic diagram of Sna3 indicating its orientation in the membrane and the locations of K19, K125, and the PPAY motif spanning amino acids 106-109. The N-terminal cytosolic domain of Sna3 consists of amino acids 1-21, and its C-terminal cytosolic domain consists of amino acids 64-133. (B) Western blot analysis of extracts prepared from wild-type cells (SEY6210), pep4 cells (TVY1), and pep4 doa4C571S cells (MWY6) transformed with empty low-copy vector (pRS416) or with pRS416 encoding Sna3-GFP (pSNA3-GFP; Katzmann et al., 2004). Asterisks indicate ubiquitinated forms of Sna3-GFP and CPS. PGK was examined as a control to indicate loading of equivalent lysate amounts. The reduced intensity of CPS in pep4 doa4C571S extracts compared with wild-type and pep4 extracts might be due to the fact that, in pep4 doa4C571S cells, some of the total pool of CPS is monoubiquitinated and some is not. (C) Western blot analysis of extracts prepared from pep4 SNA3-GFP cells (MMY161) transformed either with empty high-copy vector (pRS202) or pRS202 encoding wild-type DOA4 (pGO289; Luhtala and Odorizzi, 2004). Asterisks indicate ubiquitinated forms of Sna3-GFP. Note that the low endogenous expression of Doa4 is difficult to detect. (D) Western blot analysis of extracts prepared from pep4 doa4C571S SNA3-HA cells (MMY64) that had been transformed either with empty low-copy vector (pRS414) or pRS414 encoding Myc-Ub (pUB223; Berset et al., 2002) and incubated with 250 µM CuSO4 to induce overexpression of Myc-Ub. Unmodified full-length Sna3-HA migrates at 25 kDa. Equivalent loading of each extract was confirmed by blotting for PGK (data not shown). In B–D, antibodies used for Western blotting are indicated on the left, and the migration of molecular weight standards (kDa) are indicated on the right. Blotting of the samples shown in B with anti-Ub antibodies confirmed that Sna3-HA was polyubiquitinated. (E) Fluorescence and DIC microscopy of wild-type cells (SEY6210) and doa4C571S cells (GOY100) transformed with pSNA3-GFP or a high-copy vector encoding GFP-CPS (pGO47; Odorizzi et al., 1998). Bar, 2.5 µm.

Polyubiquitination of Sna3-GFP was not significantly affected by substitution of arginine in place of either K₁₉ or K₁₂₅ but was dramatically reduced upon simultaneous replacement of both K₁₉ and K₁₂₅ (K₀; Figure 2A). Higher molecular-weight forms of the K₀ mutant Sna3-GFP fusion protein could be faintly detected upon prolonged exposure (Figure 2A), raising the possibility that the N terminus of Sna3 can serve as an alternative site of ubiquitination (Ciechanover and Ben-Saadon, 2004). However, these data indicate that polyubiquitination of Sna3 predominantly occurs at either of its two cytosolic lysine residues.

View larger version (44K): [in this window] [in a new window]   Figure 2. Rsp5 mediates polyubiquitination at either K19 or K125 of Sna3. (A) Western blot analysis of extracts prepared from pep4 doa4C571S sna3 cells (MMY153) transformed with wild-type pSNA3-GFP or mutagenized pSNA3-GFP in which arginine was substituted for K19 (pMM158), K125 (pMM152), or both K19 and K125 (K0; pMM159). (B) Western blot analysis of extracts prepared from pep4 doa4C571S RSP5 TUL1 BSD2 cells (MMY64), pep4 doa4C571S rsp5G555D TUL1 BSD2 cells (MMY127), pep4 doa4C571S rsp5G555D tul1 BSD2 cells (MMY157), pep4 doa4C571S RSP5 tul1 BSD2 cells (GOY155), or pep4 doa4C571S RSP5 TUL1 bsd2 (GOY157) cells transformed with pSNA3-GFP. In A and B, antibodies used for Western blotting are indicated on the left, and the migration of molecular weight standards (kDa) are indicated on the right. (C) Fluorescence and DIC microscopy of bsd2 cells (MMY56) transformed with pSNA3-GFP or pGO47. Bar, 2.5 µm.

The PPxY Motif of Sna3 Mediates Direct Binding to Rsp5 WW Domains
In addition to its catalytic HECT domain, Rsp5 contains a phospholipid-binding C2 domain and three WW domains (Figure 3A). The WW domains of Rsp5 bind directly to PPxY or PxY amino acid sequences located in a variety of proteins (Chang et al., 2000). The presence of a PPxY motif in the C-terminal cytosolic domain of Sna3 (Figure 1A) raised the possibility that this sequence mediates direct association with Rsp5. Indeed, a hexahistidine (His₆)-tagged peptide corresponding to the C-terminal cytosolic domain of Sna3 (Sna3^CT) was isolated using glutathione-Sepharose affinity resin when mixed with glutathione S-transferase (GST) fused to a segment of Rsp5 that encompassed all three of its WW domains (WW123) but not when mixed with GST alone (Figure 3B). The binding between His₆-Sna3^CT and GST-WW123 was slightly decreased by mutation of P₁₀₆ in Sna3 but was more reduced by mutation of P₁₀₇ and completely eliminated by mutation of Y₁₀₉ (Figure 3B). In contrast, mutation of A₁₀₈ had no apparent effect on the binding of His₆-Sna3^CT to GST-WW123 (Figure 3C), consistent with the binding site for Rsp5 WW domains conforming to the PPxY consensus sequence.

View larger version (34K): [in this window] [in a new window]   Figure 3. The PPAY motif of Sna3 mediates direct binding to Rsp5 WW domains. (A) Schematic diagram of Rsp5 indicating the positions of its C2 domain, three WW domains, and catalytic HECT domain, including amino acid G555 important for ubiquitination of MVB cargoes (Katzmann et al., 2004). (B and C) Coomassie-stained gels containing input amounts and glutathione-Sepharose pulldowns of the indicated recombinant fusion proteins expressed in bacteria and purified by affinity isolation. Inputs correspond to 50% of the amount of each recombinant protein used for pulldowns. GST was fused to the N terminus of a segment of Rsp5 containing all three of its WW domains (WW123; pIM2). His6 was fused to a peptide corresponding to the wild-type C-terminal cytosolic domain of Sna3 (His6-Sna3CT WT; pMM143) or to the same peptide sequence containing the P106A, P107L, Y109A, A108P, or A108Q substitutions (pMM168, pMM154, pMM169, pMM204, or pMM205, respectively). In B and C, the migration of molecular-weight standards (kDa) are indicated on the left.

View larger version (70K): [in this window] [in a new window]   Figure 4. Binding of Sna3 to individual WW domains of Rsp5. Coomassie-stained gel containing glutathione-Sepharose pulldowns of the indicated recombinant fusion proteins expressed in bacteria and purified by affinity isolation. GST was fused to the N terminus of a segment of Rsp5 containing each individual wild-type WW domain (WW1, WW2, or WW3; pIM6, pIM10, or pIM8, respectively) or to each WW domain in which the WxxP motif was mutated by replacement of tryptophan with phenylalanine (W/F; pIM35, pIM36, or pIM37 for WW1, WW2, or WW3, respectively) or replacement of both tryptophan and proline with phenylalanine and alanine, respectively (W/F, P/A; pIM38, pIM39, or pIM40 for WW1, WW2, or WW3, respectively). The His6-Sna3CT are described in the legend to Figure 3. Migration of molecular weight standards (kDa) are indicated on the left.

The PPxY Motif Is Essential for Polyubiquitination and MVB Sorting of Sna3
Binding of Rsp5 to the PPxY motif in vivo is required for polyubiquitination of Sna3, as mutation of either P₁₀₇ or Y₁₀₉ eliminated Ub modification of Sna3-GFP (Figure 5A). Ubiquitination of Sna3-GFP was also greatly decreased by mutation of P₁₀₆ but could be faintly detected in longer exposures (Figure 5A), consistent with the in vitro binding studies indicating that the interaction between Rsp5 and Sna3 was reduced but not eliminated upon mutation of P₁₀₆ (Figure 3B). Notably, the PPxY motif was also required for sorting of Sna3 via the MVB pathway. As shown in Figure 5B, Sna3-GFP was not delivered into the vacuole lumen upon mutation of Y₁₀₉ and, instead, localized exclusively to punctate structures that were also labeled by DsRed fused to the FYVE domain, which binds to phosphatidylinositol 3-phosphate enriched in endosomal membranes (Burd and Emr, 1998; Gaullier et al., 1998). Previous studies showed that wild-type Sna3-GFP was similarly excluded from vacuoles and accumulated at endosomal compartments upon disruption of class E Vps protein functions (Reggiori and Pelham, 2001; Yeo et al., 2003; Katzmann et al., 2004), a condition in which sorting of all MVB cargoes is blocked (Odorizzi et al., 1998). However, mutation of P₁₀₆ in Sna3-GFP resulted in an intermediate phenotype in which a portion of the fusion protein was located in the vacuole lumen in addition to being localized to endosomes (Figure 5B), again consistent with P₁₀₆ having a less important role in MVB sorting. In fact, wild-type Sna3-GFP occasionally also colocalized with DsRed-FYVE at punctate structures in addition to being localized within the vacuole lumen (Figure 5B). Mutation of P₁₀₇, in contrast, resulted in a stronger mutant phenotype than that caused by the P₁₀₆ mutation in that Sna3-GFP was excluded from the vacuole lumen in the majority of cells (Figure 5B).

View larger version (34K): [in this window] [in a new window]   Figure 5. The PPAY motif is required for polyubiquitination and MVB sorting of Sna3-GFP. (A) Western blot analysis of extracts prepared from pep4 doa4C571S sna3 cells (MMY153) transformed with wild-type pSNA3-GFP or mutagenized pSNA3-GFP containing the P106A, P107L, or Y109A substitutions (pMM172, pMM99, or pMM171, respectively). Asterisks indicated ubiquitinated forms of Sna3-GFP. Antibodies used for blotting are indicated below each blot, and the migration of molecular weight standards (kDa) are indicated on the right. (B) Fluorescence and DIC microscopy of sna3 cells (MMY107) transformed with a high-copy vector encoding DsRed-FYVE (pRS425MET3-DsRed-FYVE; Katzmann et al., 2003) and with wild-type pSNA3-GFP or mutagenized pSNA3-GFP containing the P106A, P107L, or Y109A substitutions (pMM172, pMM99, or pMM171, respectively). Bar, 2.5 µm. (C) Fluorescence and DIC microscopy of FM 4-64–stained rsp5G555D cells (mvb326) or rsp5 cells (GW003) transformed with pSNA3-GFP. Bar, 2.5 µm. Because Rsp5 is required for oleic acid synthesis, which is essential for viability in yeast (Hoppe et al., 2000), rsp5 cells were provided supplemental oleic acid in the growth medium.

View larger version (56K): [in this window] [in a new window]   Figure 6. Rsp5 is not required for MVB biogenesis. Thin sections (70 nm) of wild-type (SEY6210), rsp5G555D (mvb326), and rsp5 (GW003) cells were examined by EM at 80 kV. White scale bars, 100 nm; black scale bars, 500 nm.

View larger version (31K): [in this window] [in a new window]   Figure 7. Tomographic analysis of MVB morphology. Two-dimensional cross sections and 3D models derived from 200-nm-thick section tomograms of wild-type (SEY6210; A and B) and rsp5G555D cells (mvb326; C and D). See corresponding Supplementary Videos. V, vacuole; N, nucleus. Scale bars, 100 nm. The lumenal vesicles in modeled MVBs are color-coded based on diameter (aqua, 18–23 nm; blue, 24–29 nm; bluish purple, 30–35 nm; purple, 36–41). (E) Distribution of vesicle diameters in wild-type and rsp5G555D cells (n = 330 and 333, respectively) normalized as percent of total. (F) Density of vesicles per unit lumenal MVB volume (nm3) in wild-type and rsp5G555D cells (p < 0.05, unpaired t test). (G) Vesicle surface area density (nm2) per unit lumenal MVB volume (nm3) in wild-type and rsp5G555D cells (p = 0.69, unpaired t test). The same data sets for wild-type and rsp5G555D cells were used to generate the graphs in E–G.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Most integral membrane proteins in yeast require monoubiquitination of their cytosolic domains in order to be sorted into the MVB pathway. Monoubiquitin is recognized as an MVB sorting signal by Ub-binding subunits of the class E Vps/ESCRT machinery (reviewed in Hicke and Dunn, 2003; Hurley and Emr, 2006). Sorting of Sna3, however, is Ub-independent because its delivery into the vacuole lumen is not impaired by substituting both of its cytosolic lysine residues with arginine (Reggiori and Pelham, 2001) or by mutations that either disable the cycle of MVB cargo ubiquitination and deubiquitination (Reggiori and Pelham, 2001; Katzmann et al., 2004) or prevent Ub binding by class E Vps proteins (Bilodeau et al., 2002). Here, we show that Sna3 circumvents the need for Ub by interacting directly with Rsp5, the E3 Ub ligase primarily responsible for monoubiquitination of yeast MVB cargoes (Blondel et al., 2004; Dunn et al., 2004; Hettema et al., 2004; Katzmann et al., 2004; Morvan et al., 2004).

Rsp5 functions in multiple Ub-dependent protein trafficking steps, including endocytosis, Golgi-to-endosome transport, and MVB cargo sorting (reviewed in Dupre et al., 2004). Recruitment of Rsp5 to endosomes is mediated in part by its C2 domain, which binds to phosphoinositides, including phosphatidylinositol 3-phosphate enriched in endosomal membranes (Dunn et al., 2004). Like Sna3, Rsp5 colocalizes with DsRed-FYVE at endosomes (Katzmann et al., 2004) and is trapped at late endosomal membranes in cells lacking functional Vps4 (Dunn et al., 2004), the ATPase that catalyzes dissociation of class E Vps proteins from endosomes (Hurley and Emr, 2006). Although the precise spatial relationship between Rsp5 and the class E Vps machinery is unknown, our observation that direct association with Rsp5 is required for sorting Sna3 into the MVB pathway suggests that Rsp5 is located in the vicinity of endosomal membrane microdomains where class E Vps proteins function in the sequestration and packaging of MVB cargoes into lumenal vesicles. The coordination of Rsp5 with class E Vps proteins is also consistent with previous work showing that sorting of Sna3 into the MVB pathway requires the class E Vps machinery (Reggiori and Pelham, 2001), indicating that Sna3 is transported via the same route used by Ub-dependent cargoes.

Other examples of Ub-independent MVB cargo sorting have been described, including the Cvt17 protein in yeast and the -opioid receptor in humans. As in the case of Sna3, class E Vps proteins are required for sorting Cvt17 and -opioid receptor via the MVB pathway (Epple et al., 2001; Tanowitz and Von Zastrow, 2002; Hislop et al., 2004), but how these cargoes gain access to the sorting machinery is unknown. Surprisingly, class E Vps proteins are not essential for sorting the human Pmel17 melanosomal protein into lumenal vesicles. Moreover, sorting of Pmel17 relies on sequences in its lumenal domain rather than its cytosolic domain (Theos et al., 2006), although the molecular basis for its recognition as an MVB cargo is unclear.

We have shown that recognition of Sna3 as an MVB cargo requires the PPAY sequence located in its C-terminal cytosolic domain, which mediates direct binding to the WW domains of Rsp5. In a parallel study (Oestreich et al., 2007), the PPAY of Sna3 was shown to be both necessary and sufficient for mediating MVB sorting when transplanted onto the cytosolic domain of a membrane protein not normally transported via the MVB pathway. PPxY or PxY motifs are common binding sites for WW domains in Rsp5 (Chang et al., 2000) and in the Nedd4 family of HECT domain E3 Ub ligases in mammalian cells, which are homologues of Rsp5 (Ingham et al., 2004). Notably, several types of enveloped viruses encode structural proteins containing PPxY motifs that bind Nedd4 and bud from infected cells by a process that is both topologically equivalent to MVB vesicle budding and is dependent on the functions of class E Vps proteins (Harty et al., 2000; Bouamr et al., 2003; Gottwein et al., 2003; Martin-Serrano et al., 2004; Vana et al., 2004; Segura-Morales et al., 2005; reviewed in Morita and Sundquist, 2004). Like Rsp5, Nedd4 proteins function in ubiquitination of cargoes sorted into the MVB pathway (Staub et al., 1997; Marchese et al., 2003), but whether ubiquitination of viral proteins by Nedd4 is directly required for packaging of viral particles is unclear (Morita and Sundquist, 2004). PPxY-containing viral proteins might be concentrated at sites of viral assembly through their direct interaction with Nedd4 proteins. Indeed, one member of the Nedd4 family stably associates with Tsg101, a subunit of ESCRT-I (Medina et al., 2005), indicating a physical link exists between the E3 Ub ligase and core MVB sorting components. Recently, Rsp5 was found to bind directly to Hse1, a subunit of the ESCRT-0 complex (Ren et al., 2006), which might explain how Sna3 bound to Rsp5 gains accesses the class E Vps/ESCRT MVB sorting machinery.

The residual polyubiquitination of Sna3-GFP detected upon mutation of both of its cytosolic lysines or upon its expression in rsp5^G555D cells leaves open the possibility that low level ubiquitination of Sna3 is sufficient for mediating MVB sorting. Oestreich et al. (2007) found that polyubiquitination of Sna3 occurred at its N terminus if both cytosolic lysines were mutated, but blocking N-terminal ubiquitination did not prevent the lysine-deficient mutant Sna3 from being sorted via the MVB pathway. Combined with our study, this result strongly suggests that polyubiquitination of Sna3 has no role in its MVB sorting but is most likely the result of its direct association with Rsp5. Indeed, most models regarding the mechanism of polyubiquitin chain assembly assume continuous association of an E3 ligase with the substrate during multiple rounds of Ub addition (Hochstrasser, 2006). In contrast, monoubiquitination can occur through transient or indirect E3-substrate interactions, as exemplified by Nedd4 in mammalian cells, which can monoubiquitinate substrates in the absence of any detectable physical interaction (Polo et al., 2002). Unlike Sna3, monoubiquitinated cargoes in yeast may encounter Rsp5 stochastically because of their colocalization at endosomal microdomains rather than rely on sequence-specific motifs that mediate direct association with Rsp5 (Dunn et al., 2004).

Recruitment of Rsp5 to sites where certain types of cargoes concentrate is mediated by Bsd2, a transmembrane protein containing a PPxY motif that binds directly to Rsp5 WW domains (Hettema et al., 2004). Bsd2 functions as an adaptor that directs Rsp5 activity toward proteins (such as CPS) that contain polar amino acids in their transmembrane domains (Hettema et al., 2004; Stimpson et al., 2006). Sna3 does not contain polar residues in either of its two transmembrane domains, consistent with our results indicating that neither its polyubiquitination nor its MVB sorting require Bsd2. Like Sna3, the PPxY motif in Bsd2 mediates its association with Rsp5 WW domains and its sorting via the MVB pathway (Hettema et al., 2004). Although the function of Sna3 is unknown, its similarity to Bsd2 suggests it could mediate recruitment of Rsp5 to sites where certain types of MVB cargoes are concentrated.

Previous observations of the Ub-independent sorting of Sna3 into the MVB pathway implied that the role of Ub is restricted to ESCRT-mediated recognition of cargoes such as CPS (Reggiori and Pelham, 2001; Bilodeau et al., 2002; Katzmann et al., 2004). 3D electron tomography, however, suggests that the Ub ligase activity of Rsp5 influences MVB vesicle formation because there was a higher frequency of smaller vesicles in rsp5^G555D cells compared with the broader distribution MVB vesicle sizes seen in wild-type cells. Although the size of an MVB vesicle might be related to the amount of cargoes it transports, the total surface area represented by lumenal vesicles was equivalent in wild-type and rsp5^G555D cells. Nevertheless, most yeast proteins require ubiquitination to be recognized as MVB cargoes (Hicke and Dunn, 2003). Future work might reveal why an equal capacity for MVB transport is maintained in rsp5^G555D cells and might also provide insight into how Ub influences the size of MVB vesicles.

	ACKNOWLEDGMENTS

 
We thank Megan Wemmer (University of Colorado, Boulder) for strain construction, Stefan Lanker (Oregon Health Sciences University) for providing pUB223, Scott Emr (University of California, San Diego) for providing pSNA3-GFP and yeast strain mvb326, and David Katzmann (Mayo Foundation, Rochester, MN) for communication of unpublished results. This work was supported by a grant to G.O. from the American Cancer Society (RSG-02-147-01-CSM). M.W.M. is supported by a training grant from the National Institutes of Health (5 T32 GM-07135). G.O. is an Arnold and Mabel Beckman Foundation Young Investigator.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-08-0663) on December 20, 2006.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Greg Odorizzi (odorizzi{at}colorado.edu)

Abbreviations used: ESCRT, endosomal sorting complex required for transport; MVB, multivesicular body; Ub, ubiquitin; Vps, vacuolar protein sorting.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Berset, C., Griac, P., Tempel, R., La Rue, J., Wittenberg, C., Lanker, S. (2002). Transferable domain in the G(1) cyclin Cln2 sufficient to switch degradation of Sic1 from the E3 ubiquitin ligase SCF(Cdc4) to SCF(Grr1). Mol. Cell. Biol 22, 4463–4476.[Abstract/Free Full Text]

Bilodeau, P. S., Urbanowski, J. L., Winistorfer, S. C., Piper, R. C. (2002). The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting. Nat. Cell Biol 4, 534–539.[Medline]

Blondel, M. O., Morvan, J., Dupre, S., Urban-Grimal, D., Haguenauer-Tsapis, R., Volland, C. (2004). Direct sorting of the yeast uracil permease to the endosomal system is controlled by uracil binding and Rsp5p-dependent ubiquitylation. Mol. Biol. Cell 15, 883–895.[Abstract/Free Full Text]

Bouamr, F., Melillo, J. A., Wang, M. Q., Nagashima, K., de Los Santos, M., Rein, A., Goff, S. P. (2003). PPPYVEPTAP motif is the late domain of human T-cell leukemia virus type 1 Gag and mediates its functional interaction with cellular proteins Nedd4 and Tsg101 [corrected]. J. Virol 77, 11882–11895.[Abstract/Free Full Text]

Burd, C. G. and Emr, S. D. (1998). Phosphatidylinositol(3)-phosphate signaling mediated by specific binding to RING FYVE domains. Mol. Cell 2, 157–162.[CrossRef][Medline]

Chang, A., Cheang, S., Espanel, X., Sudol, M. (2000). Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II. J. Biol. Chem 275, 20562–20571.[Abstract/Free Full Text]

Ciechanover, A. and Ben-Saadon, R. (2004). N-terminal ubiquitination: more protein substrates join in. Trends Cell Biol 14, 103–106.[CrossRef][Medline]

Cowles, C. R., Snyder, W. B., Burd, C. G., Emr, S. D. (1997). Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component. EMBO J 16, 2769–2782.[CrossRef][Medline]

Doyotte, A., Russell, M. R., Hopkins, C. R., Woodman, P. G. (2005). Depletion of TSG101 forms a mammalian "Class E" compartment: a multicisternal early endosome with multiple sorting defects. J. Cell Sci 118, 3003–3017.[Abstract/Free Full Text]

Dunn, R. and Hicke, L. (2001). Domains of the Rsp5 ubiquitin-protein ligase required for receptor-mediated and fluid-phase endocytosis. Mol. Biol. Cell 12, 421–435.[Abstract/Free Full Text]

Dunn, R., Klos, D. A., Adler, A. S., Hicke, L. (2004). The C2 domain of the Rsp5 ubiquitin ligase binds membrane phosphoinositides and directs ubiquitination of endosomal cargo. J. Cell Biol 165, 135–144.[Abstract/Free Full Text]

Dupre, S. and Haguenauer-Tsapis, R. (2001). Deubiquitination step in the endocytic pathway of yeast plasma membrane proteins: crucial role of Doa4p ubiquitin isopeptidase. Mol. Cell. Biol 21, 4482–4494.[Abstract/Free Full Text]

Dupre, S., Urban-Grimal, D., Haguenauer-Tsapis, R. (2004). Ubiquitin and endocytic internalization in yeast and animal cells. Biochim. Biophys. Acta 1695, 89–111.[Medline]

Epple, U. D., Suriapranata, I., Eskelinen, E. L., Thumm, M. (2001). Aut5/Cvt17p, a putative lipase essential for disintegration of autophagic bodies inside the vacuole. J. Bacteriol 183, 5942–5955.[Abstract/Free Full Text]

Futter, C. E., Pearse, A., Hewlett, L. J., Hopkins, C. R. (1996). Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. J. Cell Biol 132, 1011–1023.[Abstract/Free Full Text]

Gaullier, J. M., Simonsen, A., D'Arrigo, A., Bremnes, B., Stenmark, H., Aasland, R. (1998). FYVE fingers bind PtdIns(3)P. Nature 394, 432–433.[CrossRef][Medline]

Gottwein, E., Bodem, J., Muller, B., Schmechel, A., Zentgraf, H., Krausslich, H. G. (2003). The Mason-Pfizer monkey virus PPPY and PSAP motifs both contribute to virus release. J. Virol 77, 9474–9485.[Abstract/Free Full Text]

In: Guide to yeast genetics and molecular biology, ed. C. Guthrie and G. R. Fink. (2002). San Diego: Academic Press.

Harty, R. N., Brown, M. E., Wang, G., Huibregtse, J., Hayes, F. P. (2000). A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding. Proc. Natl. Acad. Sci. USA 97, 13871–13876.[Abstract/Free Full Text]

Hettema, E. H., Valdez-Taubas, J., Pelham, H. R. (2004). Bsd2 binds the ubiquitin ligase Rsp5 and mediates the ubiquitination of transmembrane proteins. EMBO J 23, 1279–1288.[CrossRef][Medline]

Hicke, L. and Dunn, R. (2003). Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu. Rev. Cell Dev. Biol 19, 141–172.[CrossRef][Medline]

Hislop, J. N., Marley, A., Von Zastrow, M. (2004). Role of mammalian vacuolar protein-sorting proteins in endocytic trafficking of a non-ubiquitinated G protein-coupled receptor to lysosomes. J. Biol. Chem 279, 22522–22531.[Abstract/Free Full Text]

Hitchcock, A. L., Auld, K., Gygi, S. P., Silver, P. A. (2003). A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery. Proc. Natl. Acad. Sci. USA 100, 12735–12740.[Abstract/Free Full Text]

Hochstrasser, M. (2006). Lingering mysteries of ubiquitin-chain assembly. Cell 124, 27–34.[CrossRef][Medline]

Hoppe, T., Matuschewski, K., Rape, M., Schlenker, S., Ulrich, H. D., Jentsch, S. (2000). Activation of a membrane-bound transcription factor by regulated ubiquitin/proteasome-dependent processing. Cell 102, 577–586.[CrossRef][Medline]

Horazdovsky, B. F., Busch, G. R., Emr, S. D. (1994). VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins. EMBO J 13, 1297–1309.[Medline]

Hurley, J. H. and Emr, S. D. (2006). The ESCRT complexes: structure and mechanism of a membrane-trafficking network. Annu. Rev. Biophys. Biomol. Struct 35, 277–298.[CrossRef][Medline]

Ingham, R. J., Gish, G., Pawson, T. (2004). The Nedd4 family of E3 ubiquitin ligases: functional diversity within a common modular architecture. Oncogene 23, 1972–1984.[CrossRef][Medline]

Katzmann, D. J., Babst, M., Emr, S. D. (2001). Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106, 145–155.[CrossRef][Medline]

Katzmann, D. J., Sarkar, S., Chu, T., Audhya, A., Emr, S. D. (2004). Multivesicular body sorting: ubiquitin ligase Rsp5 is required for the modification and sorting of carboxypeptidase S. Mol. Biol. Cell 15, 468–480.[Abstract/Free Full Text]

Katzmann, D. J., Stefan, C. J., Babst, M., Emr, S. D. (2003). Vps27 recruits ESCRT machinery to endosomes during MVB sorting. J. Cell Biol 162, 413–423.[Abstract/Free Full Text]

Kremer, J. R., Mastronarde, D. N., McIntosh, J. R. (1996). Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol 116, 71–76.[CrossRef][Medline]

Longtine, M. S., McKenzie, A. 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef][Medline]

Losko, S., Kopp, F., Kranz, A., Kolling, R. (2001). Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 mutant. Mol. Biol. Cell 12, 1047–1059.[Abstract/Free Full Text]

Luhtala, N. and Odorizzi, G. (2004). Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes. J. Cell Biol 166, 717–729.[Abstract/Free Full Text]

Lykke-Andersen, J., Shu, M. D., Steitz, J. A. (2000). Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon. Cell 103, 1121–1131.[CrossRef][Medline]

Macias, M. J., Wiesner, S., Sudol, M. (2002). WW and SH3 domains, two different scaffolds to recognize proline-rich ligands. FEBS Lett 513, 30–37.[CrossRef][Medline]

Marchese, A., Raiborg, C., Santini, F., Keen, J. H., Stenmark, H., Benovic, J. L. (2003). The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4. Dev. Cell 5, 709–722.[CrossRef][Medline]

Martin-Serrano, J., Perez-Caballero, D., Bieniasz, P. D. (2004). Context-dependent effects of L domains and ubiquitination on viral budding. J. Virol 78, 5554–5563.[Abstract/Free Full Text]

McIntosh, R., Nicastro, D., Mastronarde, D. (2005). New views of cells in 3D: an introduction to electron tomography. Trends Cell Biol 15, 43–51.[CrossRef][Medline]

Medina, G., Zhang, Y., Tang, Y., Gottwein, E., Vana, M. L., Bouamr, F., Leis, J., Carter, C. A. (2005). The functionally exchangeable L domains in RSV and HIV-1 Gag direct particle release through pathways linked by Tsg101. Traffic 6, 880–894.[CrossRef][Medline]

Morita, E. and Sundquist, W. I. (2004). Retrovirus budding. Annu. Rev. Cell Dev. Biol 20, 395–425.[CrossRef][Medline]

Morvan, J., Froissard, M., Haguenauer-Tsapis, R., Urban-Grimal, D. (2004). The ubiquitin ligase Rsp5p is required for modification and sorting of membrane proteins into multivesicular bodies. Traffic 5, 383–392.[CrossRef][Medline]

Mullock, B. M., Bright, N. A., Fearon, C. W., Gray, S. R., Luzio, J. P. (1998). Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent. J. Cell Biol 140, 591–601.[Abstract/Free Full Text]

Odorizzi, G., Babst, M., Emr, S. D. (1998). Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body. Cell 95, 847–858.[CrossRef][Medline]

Odorizzi, G., Katzmann, D. J., Babst, M., Audhya, A., Emr, S. D. (2003). Bro1 is an endosome-associated protein that functions in the MVB pathway in Saccharomyces cerevisiae. J. Cell Sci 116, 1893–1903.[Abstract/Free Full Text]

Oestreich, A. J., Aboian, M., Lee, J., Azmi, I., Payne, J., Issaka, R., Davies, B. A., Katzmann, D. J. (2007). Characterization of multiple multivesicular body sorting determinants within Sna3: a role for the ubiquitin ligase Rsp5. Mol. Biol. Cell 18, 707–720.[Abstract/Free Full Text]

Peng, J., Schwartz, D., Elias, J. E., Thoreen, C. C., Cheng, D., Marsischky, G., Roelofs, J., Finley, D., Gygi, S. P. (2003). A proteomics approach to understanding protein ubiquitination. Nat. Biotechnol 21, 921–926.[CrossRef][Medline]

Polo, S., Sigismund, S., Faretta, M., Guidi, M., Capua, M. R., Bossi, G., Chen, H., De Camilli, P., Di Fiore, P. P. (2002). A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins. Nature 416, 451–455.[CrossRef][Medline]

Reggiori, F. and Pelham, H. R. (2001). Sorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting. EMBO J 20, 5176–5186.[CrossRef][Medline]

Reggiori, F. and Pelham, H. R. (2002). A transmembrane ubiquitin ligase required to sort membrane proteins into multivesicular bodies. Nat. Cell Biol 4, 117–123.[CrossRef][Medline]

Ren, J., Kee, Y., Huibregtse, J. M., Piper, R. C. (2006). Hse1, a component of the yeast Hrs-STAM ubiquitin sorting complex, associates with ubiquitin peptidases and a ligase to control sorting efficiency into multivesicular bodies. Mol. Biol. Cell 18, 324–335.

Rieder, S. E., Banta, L. M., Kohrer, K., McCaffery, J. M., Emr, S. D. (1996). Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant. Mol. Biol. Cell 7, 985–999.[Abstract]

Robinson, J. S., Klionsky, D. J., Banta, L. M., Emr, S. D. (1988). Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol. Cell. Biol 8, 4936–4948.[Abstract/Free Full Text]

Sambrook, J. and Russell, D. (2001). In: Molecular cloning: a laboratory manual, Woodbury, NY: Cold Spring Harbor Laboratory Press.

Segura-Morales, C., Pescia, C., Chatellard-Causse, C., Sadoul, R., Bertrand, E., Basyuk, E. (2005). Tsg101 and Alix interact with murine leukemia virus Gag and cooperate with Nedd4 ubiquitin ligases during budding. J. Biol. Chem 280, 27004–27012.[Abstract/Free Full Text]

Sikorski, R. S. and Hieter, P. (1989). A system of shuttle vec