• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of mbcLink to Publisher's site
Mol Biol Cell. Jul 2004; 15(7): 3031–3041.
PMCID: PMC452561

Ent5p Is Required with Ent3p and Vps27p for Ubiquitin-dependent Protein Sorting into the Multivesicular Body

Jennifer Lippincott-Schwartz, Monitoring Editor

Abstract

At the late endosomes, cargoes destined for the interior of the vacuole are sorted into invaginating vesicles of the multivesicular body. Both PtdIns(3,5)P2 and ubiquitin are necessary for proper sorting of some of these cargoes. We show that Ent5p, a yeast protein of the epsin family homologous to Ent3p, localizes to endosomes and specifically binds to PtdIns(3,5)P2 via its ENTH domain. In cells lacking Ent3p and Ent5p, ubiquitin-dependent sorting of biosynthetic and endocytic cargo into the multivesicular body is disrupted, whereas other trafficking routes to the vacuole are not affected. Ent3p and Ent5p are associated with Vps27p, a FYVE domain containing protein that interacts with ubiquitinated cargoes and is required for protein sorting into the multivesicular body. Therefore, Ent3p and Ent5p are the first proteins shown to be connectors between PtdIns(3,5)P2- and the Vps27p-ubiquitin-driven sorting machinery at the multivesicular body.

INTRODUCTION

Endosomes are crossroads between the biosynthetic and the endocytic pathways. Here, proteins destined for the lysosome/vacuole are segregated away from proteins recycling back to the plasma membrane or to the Golgi apparatus. A critical membrane segregation and protein-sorting event takes place in late endosomes, regions of endosomal membranes invaginate and vesicles bud into the lumen of the organelle, giving rise to the multivesicular body (MVB; Felder et al., 1990 blue right-pointing triangle). Mature MVB fuses with the vacuole/lysosome releasing internal vesicles into its lumen and thereby exposing them to the activity of hydrolytic enzymes (Futter et al., 1996 blue right-pointing triangle; Odorizzi et al., 1998 blue right-pointing triangle). This mechanism allows a subset of endosomal membrane proteins to be sorted to the lumen of the lysosome/vacuole, whereas others are either selectively recycled back to the Golgi or to the plasma membrane, or they end up on the lysosomal/vacuolar membrane (Pelham, 2002 blue right-pointing triangle). In yeast, the MVB pathway is crucial for the regulated turnover of pheromone receptors and of some permeases (Odorizzi et al., 1998 blue right-pointing triangle). Further, biosynthetic transmembrane proteins such as carboxypeptidase S (Cps1p) and Phm5p follow this route to reach their final destination, the vacuolar lumen (Odorizzi et al., 1998 blue right-pointing triangle; Reggiori and Pelham, 2001 blue right-pointing triangle; Epple et al., 2003 blue right-pointing triangle).

Ubiquitination has been implicated as an endosomal-sorting signal. Cps1p and Phm5p ubiquitination is essential to target them into the vacuolar lumen after sorting at the MVB (Katzmann et al., 2001 blue right-pointing triangle; Reggiori and Pelham, 2001 blue right-pointing triangle). Potential cargo receptors responsible for recognition and recruitment of ubiquitinated cargo into MVB vesicles are class E Vps (vacuolar protein sorting) proteins. Class E mutants accumulate cargo destined for the vacuole in an exaggerated endosomal structure and all (currently identified) class E proteins are required for sorting at the MVB (Conibear and Stevens, 1998 blue right-pointing triangle). Vps23p, a class E protein of the ESCRT-I complex (endosomal-sorting complex required for transport), carries an ubiquitin-conjugating (UBC)-like domain and interacts directly with ubiquitin in vitro and with ubiquitinated Cps1p in vivo (Katzmann et al., 2001 blue right-pointing triangle). By binding to ubiquitinated cargo and then activating ESCRT-II, which in turn acts upstream of ESCRT-III, ESCRT-I is the first effector of a coordinated cascade necessary for ubiquitin-dependent protein sorting at the MVB (Babst et al., 2002a blue right-pointing triangle, 2002b blue right-pointing triangle). Two other class E proteins, Vps27p and Hse1p, contain ubiquitin-interacting motifs (UIMs) and bind ubiquitin in vitro (Bilodeau et al., 2002 blue right-pointing triangle; Polo et al., 2002 blue right-pointing triangle; Raiborg et al., 2002 blue right-pointing triangle; Shih et al., 2002 blue right-pointing triangle). Mutations in the UIMs of Vps27p and Hse1p cause defects in sorting of Cps1p and Ste2p, suggesting that a Vps27p/Hse1p complex is a sorting receptor for ubiquitinated proteins at the MVB. Vps27p binds PtdIns(3)P via its FYVE domain (Burd and Emr, 1998 blue right-pointing triangle; Gary et al., 1998 blue right-pointing triangle; Gaullier et al., 1998 blue right-pointing triangle; Wishart et al., 2001 blue right-pointing triangle; Burda et al., 2002 blue right-pointing triangle). PtdIns(3)P is found on the cytoplasmic side of endosomes, of internal vesicles of the MVB and of the vacuole (Gillooly et al., 2000 blue right-pointing triangle). Vps27p binds PtdIns(3)P and ubiquitin and therefore constitutes a direct link between these two sorting signals. Accordingly, recent data showed that Vps27p initiates the MVB sorting reaction and directs the recruitment of ubiquitinated cargo into MVB vesicles in association with ESCRT-I (Bilodeau et al., 2003 blue right-pointing triangle; Katzmann et al., 2003 blue right-pointing triangle).

The Ent1 to Ent5 proteins are ENTH (epsin NH2-terminal homology) domain proteins (Kay et al., 1999 blue right-pointing triangle; Wendland et al., 1999 blue right-pointing triangle; Duncan et al., 2002 blue right-pointing triangle). The ENTH domain promotes membrane recruitment by binding phosphoinositides (De Camilli et al., 2002 blue right-pointing triangle). We have recently reported that Ent3p is a specific PtdIns(3,5)P2 effector involved in protein sorting at the MVB in yeast (Friant et al., 2003 blue right-pointing triangle). Analysis of yeast mutants unable to synthesize PtdIns(3,5)P2, such as fab1, vac7, and vac14, revealed that this phospholipid is also required for sorting ubiquitinated cargoes into the MVB (Bonangelino et al., 1997 blue right-pointing triangle; Odorizzi et al., 1998 blue right-pointing triangle; Reggiori and Pelham, 2001 blue right-pointing triangle; Dove et al., 2002 blue right-pointing triangle).

In this study we investigate the role of Ent5p that also specifically binds to PtdIns(3,5)P2. It is associated with the ubiquitin-binding protein Vps27p and together with Ent3p is required for ubiquitin-dependent protein sorting into the MVB.

MATERIALS AND METHODS

Plasmids and Yeast Strains

Plasmids used in this study are listed in Table 1. Yeast strains used in this study are RH2964 (WT), FLY673 1b (Mata bar1 ura3 leu2 trp1 ade2 ent3::HIS3), FLY674 1a (Mata bar1 ura3 leu2 trp1 ade2 ent4::KanMX), FLY675 1b (Mata bar1 ura3 leu2 trp1 ade2 ent5::HIS3), FLY676 3d (Mata bar1 ura3 leu2 trp1 ade2 ent4::KanMX ent5::HIS3), FLY678 2b (Mata bar1 ura3 leu2 trp1 ade2 ent3::HIS3 ent4::KanMX), FLY680 16a (Mata bar1 ura3 leu2 trp1 ade2 ent3::HIS3 ent5::HIS3), BY4272 (WT), vps24Δ, vsp36Δ, vps37Δ, PLY1020 (WT), PLY2857 (Matα pep4-3 ura3-52 leu2-3 his4-519 ade6 vps27::KanMX), PLY2784 (Matα pep4-3 ura3-52 leu2-3 his4-519 ade6 vps27::KanMX hse1::KanMX), vps24Δvps27Δ (Mata ura3 leu2 vps24::KanMX vps27::KanMX), FY833 (WT), and FY833-fab1Δ1 (fab1Δ).

Table 1.
Plasmids used in this study

Endocytosis Assay and Quinacrine Staining

Lucifer yellow (LY) uptake and quinacrine assays were performed as previously described (Friant et al., 2003 blue right-pointing triangle).

Lipid Dot Blot Assay

Lipid solutions containing 100 pmol/μl phospholipids dissolved in CHCl3/MetOH/HCl (50:50:0.1 vol/vol) were spotted on a nitrocellulose membrane and allowed to dry for 1 h. The membrane was blocked in 3% fatty acid free BSA in TBS-Tween for 1 h and incubated overnight with the GST-ENTH domain of Ent5p (at 0.3 μg/ml). After washing, protein binding was revealed with anti-GST.

Liposome Recruitment Assay

Recruitment reactions were performed as previously described (Friant et al., 2003 blue right-pointing triangle). The FYVE domain was a kind gift of M.A. Lemmon.

Subcellular Fractionation, Western Blot, Gel filtration, Coimmunoprecipitation, and Membrane Fractionation

Subcellular fractionation was carried out as described in Bonangelino et al. (1997 blue right-pointing triangle). Mouse monoclonal anti-GFP (Roche, Nutley, NJ), anti-PGK, and anti-Vph1p antibodies (Molecular Probes, Eugene, OR), anti-HA (Roche Diagnostics Corp.) and anti-GST (Sigma-Aldrich, St. Louis, MO), rabbit anti-Emp47 (kind gift from R. Duden), and anti-Ent3 antibodies (Friant et al., 2003 blue right-pointing triangle) were used. For detection of His6-tagged proteins the SuperSignal West HisProbe Kit from Pierce (Rockford, IL) was used. For gel filtration, cytosol was prepared by glass bead lysis in PBS containing 0.25 M sorbitol, protease inhibitor cocktail (Sigma), and PMSF, followed by a 10-min spin at 13 000 rpm and a 30-min spin at 100,000 × g. Five to 9 mg protein was injected on a Superose 6 column (Pharmacia, Arlington Heights, IL), and 10 μg protein/fraction was analyzed by Western blot. Yeast cytosol or fractions from gel filtration were adjusted to identical protein amounts and immunoprecipitated with Ent3p antibodies and then subjected to anti-HA or anti-GFP Western blotting. Membrane fractionations on sucrose density gradients were carried out as described (Holthuis et al., 1998 blue right-pointing triangle). Mouse Pep12p and Vps10p antibodies from Molecular Probes were used for detection of endosomal fractions.

Pull-down Experiments and Ubiquitin-binding Assays

GST-pull down experiments were carried out as described (Cosson and Letourneur, 1994 blue right-pointing triangle), but yeast cells were lysed in PBS, 10 mM EDTA, 0.5% Triton, protease inhibitor cocktail, and PMSF. For binding to ubiquitin-sepharose, 50 μl ubiquitin-sepharose (Sigma) was incubated with 20 μg recombinant protein for 4 h, and washed in TBST as described (Aguilar et al., 2003 blue right-pointing triangle).

Immunoprecipitations and Metabolic Labeling

Metabolic labeling, pulse-chase analyses, and immunoprecipitations were performed as previously described, by using mouse anti-ALP (Molecular Probes) and rat anti-HA antibodies (Sigma) and protein A-Sepharose (Amersham Biosciences Europe GmbH, Freiburg, Germany; Friant et al., 2003 blue right-pointing triangle). For immunoprecipitation of ubiquitinated proteins, cells overexpressing ubiquitin (necessary to allow detection of a signal) and expressing GFP-Cps1 were grown to an OD600 = 0.5, precipitated in 10% TCA, washed in acetone, resuspended in 200 μl TE, protease inhibitor cocktail, PMSF, and 5 mM NEM, and vortexed for 5 min with glass beads. Thorner buffer, 200 μl, was added and broken cells were incubated at 50°C for 10 min. After centrifugation for 10 min at 13,000 rpm, 100 μl of supernatant was subjected to immunoprecipitation with ant-GFP antibody (Roche). Immunoprecipitated proteins were revealed with anti-ubiquitin antibody (Zymed, San Francisco, CA).

Microscopy

Living cells expressing GFP-Cps1, GFP-Phm5, GFP-DPAP B, or GFP-Ent5 were harvested at an OD600 of 0.5 and resuspended in PBS before visualization. In vivo labeling with FM4-64 was done as described (Vida and Emr, 1995 blue right-pointing triangle). Visualization of cells was performed on a fluorescence Zeiss Axioplan II (Thornwood, NY) or Nikon Optiphot-2 microscope (Melville, NY) equipped with FITC and rhodamine filters, and Nomarski optics. Images were captured with a CoolSnap FX Roper Scientific (Tuscon, AZ) or a Photonic Science digital camera (Turnbridge Wells, UK).

Computer Analysis

Sequence alignments were done using the ClustalW program (Thompson et al., 1994 blue right-pointing triangle) and displayed with ESPript (Gouet et al., 1999 blue right-pointing triangle). Database searches were performed using BLAST and ψ BLAST servers at NIH.

RESULTS

Ent5p Is Redundant with Ent3p and Not Required for the Internalization Step of Endocytosis

We have recently characterized ent3-1, a temperature-sensitive mutant defective in protein sorting at the MVB (Friant et al., 2003 blue right-pointing triangle). The ent3Δ deletion mutant did not display the same phenotype as the ent3-1 mutant, suggesting that the yeast genome encoded a functional homologue. There are five different yeast ENTH domain-containing proteins named Ent1p to Ent5p (Figure 1). In contrast to Ent3p, Ent1p and Ent2p were shown to be required for endocytosis (Wendland et al., 1999 blue right-pointing triangle). The function of Ent4p is not known, and Ent5p was recently identified as a functional homologue of Ent3p, involved in clathrin-mediated trafficking at the late Golgi (Duncan et al., 2002 blue right-pointing triangle). The single ent3Δ, ent4Δ, and ent5Δ and the double ent3Δent4Δ and ent4Δent5Δ deletion strains displayed no obvious phenotype, but the double ent3Δent5Δ mutant was cold sensitive for growth on YPD plates (unpublished data) and showed highly fragmented vacuoles (see also Figure 4, A and D, and and5A).5A). This vacuolar phenotype had already been observed in ent3-1 mutant cells (Friant et al., 2003 blue right-pointing triangle). These results suggested that Ent5p and Ent3p shared functions in MVB sorting, in the light of what has been previously shown for clathrin-mediated membrane trafficking (Duncan et al., 2002 blue right-pointing triangle; Friant et al., 2003 blue right-pointing triangle). The ENTH domain is a membrane-interacting module found in a number of proteins acting at early stages of endocytosis (De Camilli et al., 2002 blue right-pointing triangle). We therefore asked whether the presence of Ent5p was required with Ent3p for the internalization step of endocytosis. Uptake of lucifer yellow (LY) was analyzed in ent3Δ, ent5Δ, and ent3Δent5Δ mutant cells and they accumulated LY in the vacuole like wild-type cells (unpublished data). These data show that Ent5p, like its homologue Ent3p, serves a function different from the “classical” role of epsins in endocytosis.

Figure 1.
Sequence alignment of the ENTH domains of yeast epsins. The sequence of the rat epsin1 was aligned to sequences from yeast Ent1p, Ent2p, Ent3p, Ent4p, and Ent5p proteins. Secondary structure of rat epsin1 is shown above the alignment. Conserved residues ...
Figure 4.
Ent3p and Ent5p are required for sorting of ubiquitinated biosynthetic and endocytic proteins to the MVB. (A) Wild-type (RH2964) and ent3Δent5Δ (FLY680 16a) cells were transformed with pGFP-Cps1 or with pGFP-Phm5, labeled with FM4-64 and ...
Figure 5.
Ent3p and Ent5p neither required for sorting of DPAP B to the vacuolar membrane nor for acidification of the vacuole. (A) Wild-type (RH2964) and ent3Δent5Δ (FLY680 16a) cells were transformed with pGFP-DPAP B and cells were observed by ...

Ent5p-GFP Localizes to Endosomal Structures

To analyze the intracellular localization of Ent5p, we fused green-fluorescent protein (GFP) to the NH2-terminus of full-length Ent5p. This fusion protein is functional, as it complements the fragmented vacuolar phenotype of ent3Δent5Δ mutant cells. In wild-type cells, GFP-Ent5p localized to the cytoplasm and to punctate structures, indicating association with endosomal compartments (Figure 2A). Some of the dots colocalized with the staining obtained after a short pulse with FM4-64, which visualizes endosomal structures (Figure 2B; Vida and Emr, 1995 blue right-pointing triangle). In addition, GFP-Ent5p was introduced into class E vps mutants that accumulate enlarged prevacuolar/endosomal structures, vps27Δ and vps24Δ, which affects the ESCRT-III complex (Figure 2B). In these class E vps mutants, the majority of GFP-Ent5p was localized to the class E compartment, as seen by its colocalization with FM4-64 (Figure 2B). In the vps24Δvps27Δ double mutant, GFP-Ent5p was also localized to the class E compartment (Figure 2B), thus indicating that Vps27p is not responsible the localization of Ent5p to the class E compartment of vps24Δ cells. The intracellular localization of GFP-Ent5p was further characterized by a differential sedimentation assay. In wild-type cells, the majority of GFP-Ent5p was membrane-associated in the fraction that pelleted at 13,000 × g (P13) that contains large organelles such as vacuoles, ER, nuclei, or structures like the cytoskeleton, and in the high-speed membrane fraction (P100) containing Golgi membranes, endosomes, and vesicles, but also for a minor part in the cytoplasmic pool (S100; Marcusson et al., 1994 blue right-pointing triangle). A similar fractionation pattern for GFP-Ent5p was observed in the class E mutants vps23Δ or vps27Δ (unpublished data).

Figure 2.
GFP-Ent5 localizes to endosomal structures. (A) Wild-type cells (FY833) and fab1Δ1 expressing GFP-Ent5 (pFL644) were observed by fluorescent and Nomarski microscopy. (B) Wild-type cells (BY4741), vps24Δ, vps27Δ, and vps24Δ ...

The intracellular distribution of GFP-Ent5p was also analyzed by subcellular fractionation on an equilibrium sucrose density gradient. As shown in Figure 2C, GFP-Ent5p fractionated away from the vacuolar marker ALP and most closely resembled the distribution of Pep12p, the t-SNARE for a late endosomal/prevacuolar compartment and of Vps10p, the early endosomal marker (Figure 2C). All these results suggest that Ent5p is a cytosolic protein able to associate with membranes and is found predominantly on endosomal structures.

Ent5p Binds Specifically to PtdIns(3,5)P2

The ENTH domain is a phospholipid binding module (De Camilli et al., 2002 blue right-pointing triangle). We have recently shown that the ENTH domain of Ent3p specifically binds PtdIns(3,5)P2 in vivo and in vitro (Friant et al., 2003 blue right-pointing triangle). To determine the lipid specificity of Ent5p, liposome recruitment assays with recombinant GST-Ent5 fusions were performed. GST-Ent5 or GST-ENTH domain were cosedimented with PtdIns(3,5)P2 containing liposomes but not with liposomes containing PtdIns(3)P, PtdIns(3,4)P2, PtdIns(4,5)P2, or PtdIns(4)P (Figure 3A). In contrast, a truncation of Ent5p lacking the ENTH domain was unable to interact with any of the liposomes tested, thus demonstrating that the ability of Ent5p to bind PtdIns(3,5)P2 was dependent on its ENTH domain (unpublished data). As a control, we tested the lipid specificity of the FYVE domain of Hrs-1 in our liposome recruitment assay (Figure 3A), and the FYVE domain bound specifically to PtdIns(3)P as previously reported (Sankaran et al., 2001 blue right-pointing triangle). As can be seen in Figures Figures3A3A and and6B,6B, recombinant GST-Ent5 consistently appeared as a triplet. To date we are unable to discern the nature of or the modifications on the different species observed. We also performed a protein-lipid overlay assay using the ENTH domain of Ent5p. This assay confirmed the results of the liposome recruitment assay, in that the ENTH domain of Ent5p bound only to PtdIns(3,5)P2 (Figure 3B).

Figure 3.
Ent5p binds specifically to PtdIns(3,5)P2. (A) GST-Ent5 or GST-ENTH of Ent5p were incubated with either PC (control) or PC-based liposomes containing 5% (mol/mol) PtdIns(3,5)P2, PtdIns(3,4)P2, PtdIns(4,5)P2, PtdIns(3)P, or PtdIns(4)P. After centrifugation, ...
Figure 6.
Ent3p and Ent5p are not required for cargo ubiquitination and are unable to bind ubiquitin. (A) Wild-type (RH2964) and ent3Δent5Δ (FLY680 16a) cells expressing pGFP-Cps1 and pKN32 (2μ ubiquitin) were lysed, lysates were subjected ...

To determine whether lack of PtdIns(3,5)P2 affects the intracellular localization of Ent5p, fab1Δ cells that are impaired in PtdIns(3,5)P2 synthesis were transformed with the GFP-Ent5p construct and analyzed by fluorescent microscopy (Figure 2A). GFP-Ent5p was completely diffuse in the cytosol of fab1Δ cells. This suggests that Ent5p localizes to endosomes by virtue of its binding to PtdIns(3,5)P2. Taken together, our data demonstrate that Ent5p specifically binds to PtdIns(3,5)P2 via its ENTH domain and show that Ent5p is a new PtdIns(3,5)P2 effector in yeast.

Ent5p, together with Ent3p, Is Specifically Required for Ubiquitin-dependent Sorting of Biosynthetic and Endocytic Cargoes into the MVB

It is well established that the lipid kinase Fab1p and its product PtdIns(3,5)P2 are required for the correct sorting of ubiquitinated biosynthetic and endocytic cargo proteins at the MVB (Odorizzi et al., 1998 blue right-pointing triangle; Reggiori and Pelham, 2001 blue right-pointing triangle; Shaw et al., 2003 blue right-pointing triangle). Here we identified Ent5p as a new PtdIns(3,5)P2 effector in yeast, redundant with Ent3p. Both proteins are localized to endosomes, the site of MVB formation (Friant et al., 2003 blue right-pointing triangle). We therefore asked whether Ent3p and Ent5p were also implicated in ubiquitin-dependent MVB sorting and analyzed the intracellular localization of GFP-Cps1 and GFP-Phm5, two ubiquitinated biosynthetic cargo proteins, in ent3Δ, ent5Δ, and ent3Δent5Δ mutant cells (Figure 4). Precursor forms of Cps1p and Phm5p are synthesized as type II transmembrane proteins. After transit through the secretory pathway to endosomes, they are sorted into invaginating MVB vesicles, resulting in exposure of their short cytoplasmic N-terminal domain to the lumen of the vesicles. Delivery of the vesicles to the vacuolar lumen allows hydrolytic clipping of the precursors to their mature forms: the large C-terminal part is released from the transmembrane domain into the vacuolar lumen where it exerts its activity. In wild-type, ent3Δ, and ent5Δ cells, expression of GFP-Cps1 or GFP-Phm5 gave rise to a clear staining of the vacuolar lumen (Figure 4A and unpublished data). In contrast, in ent3Δent5Δ mutant cells, GFP-Cps1 and GFP-Phm5 localized to the fragmented vacuolar membrane and not to the vacuolar lumen, as shown by colocalization with the dye FM4-64 (Figure 4A). To confirm that Ent3p and Ent5p were both required for sorting of GFP-Phm5 and GFP-Cps1 at the MVB, total yeast cell extracts were analyzed by Western blotting and probed with an anti-GFP antibody (Figure 4, B and C). In wild-type, ent3Δ, and ent5Δ cells, free GFP released by vacuolar enzymes after maturation of GFP-Cps1 and GFP-Phm5 were readily detected. In contrast, free GFP was absent from ent3Δent5Δ mutant cells, suggesting that GFP-Phm5 and GFP-Cps1 had not been exposed to the activity of vacuolar proteases and therefore had not been properly sorted into the MVB pathway.

The endocytic cargo Ste2p is also ubiquitinated and this modification is requisite for its sorting into internal vesicles of the MVB as well as for its internalization at the plasma membrane (Katzmann et al., 2001 blue right-pointing triangle; Bilodeau et al., 2002 blue right-pointing triangle; Shih et al., 2002 blue right-pointing triangle). To find out whether Ent3p and Ent5p were also required for the vacuolar targeting of ubiquitin-modified endocytic cargo, the intracellular localization of GFP-Ste2 was analyzed by fluorescence microscopy (Figure 4D). In wild-type cells, GFP-Ste2 was concentrated in the vacuolar lumen. In ent3Δent5Δ mutant cells, GFP-Ste2 was mainly localized to the vacuolar membrane, with some residual staining in the vacuolar lumen, thus suggesting that the correct and efficient sorting of some endocytic cargoes also requires Ent3p and Ent5p.

To ascertain whether the sorting defect observed in ent3Δent5Δ mutant cells was specific for transmembrane proteins sorted into internal MVB vesicles and whether sorting of proteins of the vacuolar membrane was disturbed, the intracellular localization of GFP-DPAP B (Dipeptidyl aminopeptidase B) was analyzed in these cells. DPAP B is a type II transmembrane protein that travels along the CPY pathway to the limiting membrane of the vacuole (Piper et al., 1997 blue right-pointing triangle). In wild-type as in ent3Δent5Δ mutant cells, GFP-DPAP B was found on vacuolar membranes (Figure 5A), indicating that this pathway functions in the absence of Ent3p and Ent5p.

We also followed the transport/processing of CPY and ALP by pulse-chase analysis. In wild-type cells, after a pulse with Met/Cys 35S label, the precursor forms of CPY (p1CPY and p2CPY) and ALP (proALP) can be detected and after chase of increasing time length, increasing amounts of mature CPY and ALP appear, resulting from the transport of the precursor forms to the vacuole. In ent3Δ, ent5Δ, and ent3Δent5Δ cells, ALP and CPY were correctly processed/transported, although ent3Δent5Δ mutant cells displayed a slight delay when compared with wild-type cells (unpublished data).

Fab1p, the PtdIns(3)P 5-kinase is required for proper vacuole acidification (Bonangelino et al., 1997 blue right-pointing triangle). We therefore tested the acidification of the vacuole in cells lacking Ent3p and/or Ent5p by quinacrine staining. The lumen of the vacuoles of ent3Δ, ent5Δ, or ent3Δent5Δ mutant cells showed wild-type staining of quinacrine, whereas in fab1Δ cells there is no quinacrine staining of the vacuolar lumen (unpublished data and Figure 5B).

Taken together, all these data show that the two PtdIns(3,5)P2 effectors, Ent3p and Ent5p, are specifically required for ubiquitin-dependent sorting of endocytic and biosynthetic cargo at the MVB, whereas other routes to the vacuole are not affected, and acidification of the vacuole is normal.

Ent3p and Ent5p Do Not Directly Bind to Ubiquitin

Both Ent3p and Ent5p are required for the correct sorting of ubiquitinated proteins into the MVB. To distinguish whether Ent3p and Ent5p were required for the sorting step per se or rather to mark proteins with ubiquitin for internalization, we investigated whether MVB cargo proteins themselves were ubiquitinated in cells lacking Ent3p and Ent5p. Wild-type and ent3Δent5Δ cells carrying GFP-Cps1 and a high copy number plasmid expressing ubiquitin were lysed, and cell extracts were subjected to immunoprecipitation with an anti-GFP antibody. Western blot analysis of the immunoprecipitated proteins with an anti-ubiquitin antibody revealed the presence of ubiquitinated forms of Cps1p in ent3Δent5Δ cells (Figure 6A), whereas these forms of CPS were difficult to detect in wild-type cells, as already reported previously (Katzmann et al., 2001 blue right-pointing triangle; Shih et al., 2002 blue right-pointing triangle). This result indicates that Ent3p and Ent5p function downstream of cargo ubiquitination in the sorting function.

Epsins and Ent1p and Ent2p contain two or three copies of an UIM immediately after the ENTH domain (Polo et al., 2002 blue right-pointing triangle). The function of these UIMs is to bind ubiquitinated cargo (De Camilli et al., 2002 blue right-pointing triangle; Shih et al., 2002 blue right-pointing triangle; Aguilar et al., 2003 blue right-pointing triangle). Ent3p and Ent5p are required for ubiquitin-dependent sorting at the MVB, but they do not possess obvious ubiquitin-binding domains (UBC-like, UIM, UBA, or CUE; Hofmann and Bucher, 1996 blue right-pointing triangle; Hofmann and Falquet, 2001 blue right-pointing triangle; Polo et al., 2002 blue right-pointing triangle; Shih et al., 2002 blue right-pointing triangle). To ascertain that in Ent3p and Ent5p there was no other, as yet unidentified ubiquitin-binding motif, we performed ubiquitin-binding assays. GST-Ent3 and GST-Ent5 recombinant proteins were unable to bind ubiquitin-sepharose, whereas recombinant His6-tagged Vps27p, which contains two UIMs, specifically bound to ubiquitin-sepharose (Figure 6B). Taken together, these data suggest that Ent3p and Ent5p, although necessary for sorting ubiquitinated cargo at the MVB, are required neither for ubiquitin addition to cargo proteins nor for the direct binding to ubiquitinated cargoes. Therefore, they might act via interaction with other proteins of the sorting machinery present on endosomes during MVB formation, which in turn directly interact with ubiquitinated cargo.

Ent3p, Ent5p, and Vps27p Associate

In yeast, Ent1p/Ent2p UIMs are important for the internalization of receptors at the plasma membrane, and Vps27p/Hse1p UIMs are necessary to sort biosynthetic and endocytic cargo into the MVB (Bilodeau et al., 2002 blue right-pointing triangle; Shih et al., 2002 blue right-pointing triangle). In contrast to Vps27p and Ent1p/Ent2p, Ent3p and Ent5p have no known ubiquitin-binding motif. We show that these proteins do not directly interact with ubiquitin, but are required for ubiquitin-dependent sorting at the MVB. Thus there must be (an) additional effector(s) associated with Ent3p/Ent5p to link them to ubiquitin. Since Vps27p contains UIMs and is at the top of the ESCRT cascade, it was the most obvious candidate connector between Ent3p/Ent5p and ubiquitin (Katzmann et al., 2003 blue right-pointing triangle). To investigate whether Ent3p and Ent5p interacted with Vps27p, we performed pull-down assays using recombinant GST-Ent5 and cytosol from yeast cells expressing epitope-tagged Vps27p (Vps27-HA) as the sole source of Vps27p. Vps27p and Ent3p interacted with GST-Ent5 but not with GST alone (Figure 7A). These interactions were not dependent on the presence of Hse1p, a protein associated in a complex with Vps27p (Bilodeau et al., 2002 blue right-pointing triangle), as demonstrated by the same experiment using cytosol from a strain lacking Hse1p (Figure 7A). Thus, Vps27p, without its partner Hse1p, is sufficient to promote interaction with Ent3p and Ent5p proteins.

Figure 7.
Ent3p and Ent5p are associated to the ubiquitin-binding protein Vps27p. (A) Lysates from vps27Δ (PLY2857) or vps27Δhse1Δ cells (PLY2784) expressing an HA-tagged version of Vps27p were incubated with GST-Ent5 or GST alone coupled ...

We next wanted to know whether Ent3p, Ent5p, and Vps27p associated in vivo and therefore analyzed yeast cytosol by gel filtration and coimmunoprecipitation assays. Vps27-HA expressed as the sole copy of Vps27p in vps27Δ cells eluted in multiple peaks in high-molecular-weight fractions. Four different peaks ranging from 1100 to 500 kDa were observed in fraction 1, fractions 4-6, fraction 9, and fractions 11-12 (Figure 7B). Ent3p eluted in a broad peak (fractions 4-11), with the majority of the protein found in fraction 9 (Figure 7B). Accordingly, in ent5Δ cells expressing GFP-Ent5, Ent5p coeluted with Ent3p in corresponding high-molecular-weight fractions (fractions 6-11), both proteins concentrating in a peak in fractions 7-9. To determine whether Ent5p and Vps27p are associated with Ent3p in whole cell extracts, we carried out coimmunoprecipitation assays between Ent3p and epitope-tagged Vps27p (Vps27-HA) or Ent5p (GFP-Ent5). Vps27-HA coprecipitated with Ent3p from total cytosol or from fractions of gel filtration experiments (as shown above; Figure 7C). The greatest amount of Vps27-HA coimmunoprecipitated with Ent3p from fractions corresponding to shared peaks, for example from fractions 10-12 (Figure 7C) or from fraction 9 (unpublished data), when compared with coimmunoprecipitation from other fractions, as shown here for fractions 7-8. Ent5-GFP was also coprecipitated with Ent3p from ent5Δ cytosol (Figure 7D). These experiments show that Ent3p, Ent5p, and Vps27p are associated in vivo.

Further information about Ent3p, Ent5p, and Vps27p association in vivo was obtained by gel filtration of cytosol of yeast cells deleted for Vps27p and carrying GFP-Ent5p. As shown in Figure 7B, GFP-Ent5p was almost completely depleted from the high-molecular-weight peak seen in wild-type extracts. Only a minor fraction of Ent5p remained distributed over fractions 1-10; additionally, a second peak containing the majority of the protein was seen around 230-300 kDa (fractions 16-20). The high-molecular-weight fractions containing Ent3p were shifted to even higher molecular weight fractions (fractions 1-7). This result shows that the deletion of Vps27p had a dramatic effect on the gel filtration elution profile of both Ent3p and Ent5p. On the contrary, deletion of Ent5p affected the fractionation profile of neither Vps27p nor Ent3p (unpublished data), suggesting that in the absence of Ent5p, Ent3p, and Vps27p could still be incorporated into complexes. Taken together, these data suggest that Ent3p, Ent5p, and Vps27p are found together in high-molecular-weight complexes in vivo and suggest that this association may provide a direct link between the ubiquitin- and the PtdIns(3,5)P2-dependent sorting mechanisms into the MVB.

DISCUSSION

We have recently described Ent3p, the first specific PtdIns(3,5)P2 effector required for protein sorting into the MVB in yeast (Friant et al., 2003 blue right-pointing triangle). A single ent3Δ deletion strain does not display the defects in MVB sorting observed in ent3-1 mutant cells. This suggested that the point mutation in the ENTH domain of Ent3p found in ent3-1 mutant cells had a dominant effect on MVB protein sorting and that there could be additional PtdIns(3,5)P2 effectors required for MVB sorting (Friant et al., 2003 blue right-pointing triangle). In our query for ENTH domain proteins redundant to Ent3p, we identified Ent5p. Ent5p was recently identified as a functional homologue of Ent3p associated with clathrin and Gga2p, required for efficient CPY protein trafficking at the late Golgi, suggesting that these two yeast ENTH proteins function together (Duncan et al., 2002 blue right-pointing triangle). These results and the results we describe suggest that Ent3p and Ent5p share essential functions for membrane trafficking between the late Golgi and the vacuole.

There are two sequence motifs that constitute the homology between Ent3p and Ent5p: first, the signature of the epsin family proteins, the ENTH domain, and second, the presence of several γ-ear-binding acidic motifs (Duncan et al., 2002 blue right-pointing triangle). Based on the presence of a lysine-rich ANTH-like (AP180 N-terminal homology) motif in the ENTH domain of Ent5p, but absent from the ENTH domains of Ent3p or epsin1, it has recently been proposed that the ENTH domain of Ent5p rather resembles an ANTH domain (Duncan and Payne, 2003 blue right-pointing triangle). The authors therefore suggested that the ENTH domains of Ent3p and Ent5p are functionally different. Here, we show that both ENTH domains are specific PtdIns(3,5)P2-binding modules required for protein sorting into the MVB. Despite their sequence divergence the two domains share the same lipid specificity. This could be either due to sequence or structure conservation of the critical PtdIns(3,5)P2 recognition site, or to both. It will be very interesting to gain structural, and hence mechanistic information on the two ENTH domains of Ent3p and Ent5p, and especially to identify the residues responsible for PtdIns(3,5)P2 binding.

Synthesis of PtdIns(3,5)P2 by Fab1p is required for the selective sorting of ubiquitinated cargo into the MVB (Odorizzi et al., 1998 blue right-pointing triangle; Reggiori and Pelham, 2001 blue right-pointing triangle; Shaw et al., 2003 blue right-pointing triangle). Our experiments show that the PtdIns(3,5)P2 effectors Ent3p and Ent5p are also required for the correct sorting of ubiquitinated cargo into the MVB. We show that in the absence of both proteins, biosynthetic and endocytic cargo destined to the vacuolar lumen, like Phm5p, Cps1p, and Ste2p, are mis-sorted to the vacuolar membrane, showing that Ent3p and Ent5p are required for sorting of ubiquitinated cargo into the MVB. Furthermore, we demonstrate that Ent3p and Ent5p are not necessary to mark proteins for ubiquitination and do not directly interact with ubiquitinated proteins to recruit them into vesicles. Collectively, these results strongly suggest that the PtdIns(3,5)P2 effectors Ent3p and Ent5p are required neither for ubiquitination, nor for direct recognition of ubiquitinated cargoes at the late endosome, but rather for a later step of ubiquitin-dependent protein sorting into the MVB.

Here we show that Ent3p, Ent5p, and Vps27p are associated in vivo. Our GST pull-down, gel filtration, and coimmunoprecipitation experiments suggest the three proteins to be in complexes of very high molecular weight. The very large peak observed for all three proteins (ranging from 500 to far >750 kDa) suggests that they are present in different complexes. Furthermore, the presence of the three proteins in complexes eluting in fraction 9 and the pull-down of both Vps27p and Ent3p with GST-Ent5p, suggest that they are associated with each other in this complex or that Ent3p and/or Ent5p may interact indirectly with Vps27p via the ESCRT-I complex (Bilodeau et al., 2003 blue right-pointing triangle; Katzmann et al., 2003 blue right-pointing triangle). In the absence of Vps27p, the distribution of Ent5p and Ent3p over the fractionation profile is dramatically altered. Ent5p is mainly found in lower-molecular-weight fractions, in a peak that contains subcomplexes lacking Vps27p, whereas Ent3p is found in higher-molecular-weight fractions. Moreover, the absence of Ent5p affected the fractionation profile of neither Vps27p nor Ent3p (unpublished data), suggesting that in the absence of Ent5p, Ent3p, and Vps27p could still be incorporated into complexes. These results suggest that Ent3p and Vps27p are the key effectors in these high-molecular-weight complexes, and Ent5p might be an accessory protein redundant to Ent3p. This might explain why the ent3-1 and ent3-2 mutant strains have a dominant effect on MVB protein sorting and why a double deletion of ENT3 and ENT5 is required to obtain the same phenotype as displayed by these mutants. Alternatively, complexes might exist containing either Ent3p or Ent5p together with Vps27p, and different “subcomplexes” might well transiently associate with a larger complex. Therefore, it will be interesting to determine the additional components of complexes containing Vps27p, Ent3p, and/or Ent5p and to determine the domains on Ent3p and Ent5p responsible for interaction with Vps27p.

Vps27p is one of the key effectors required for protein sorting into the MVB pathway, because it binds ubiquitin in vitro and because mutations in the UIMs of Vps27p cause defects in protein sorting into the MVB (Bilodeau et al., 2002 blue right-pointing triangle; Shih et al., 2002 blue right-pointing triangle). Furthermore, it was recently shown that Vps27p is directly recruited to PtdIns(3)P-containing endosomes where it binds to ubiquitinated MVB cargoes and subsequently recruits the ESCRT-I complex (Bilodeau et al., 2003 blue right-pointing triangle; Katzmann et al., 2003 blue right-pointing triangle). This results in sorting of ubiquitinated cargo into the MVB vesicles. Recent studies permitted the unraveling of different steps leading to the recruitment of ubiquitinated cargo to the site of MVB formation. Despite all these new data, some clues are still missing. For example, the mechanism triggering the formation of invaginating vesicles of the MVB compartment is still unknown. Here we identified two new partners of Vps27p: Ent3p and Ent5p, two PtdIns(3,5)P2 effectors required for ubiquitin-dependent protein sorting into the MVB. It has been proposed that a function of the epsin1 ENTH domain at the plasma membrane is to induce membrane curvature (Ford et al., 2002 blue right-pointing triangle). Once bound to PtdIns(4,5)P2, the 8-helix-folded ENTH domain of epsin 1 rearranges and an additional α helix is formed (α0, Figure 1). By inserting α0 into the cytoplasmic leaflet of the membrane bilayer, the ENTH domain is capable of pushing aside lipid head groups, thereby inducing curvature of the membrane and facilitating formation of clathrin-coated pits. We envisage that the role of the ENTH domains of Ent3p and Ent5p could be similar to that of epsin1. Ent3p and Ent5p, by inserting their ENTH domains into the endosomal membrane after PtdIns(3,5)P2 binding, could well induce the invagination required for the formation of internal vesicles. This process is topologically reversed when compared with formation of coated pits at the plasma membrane, and it therefore infers that the way the ENTH domains of Ent3p and Ent5p deform membranes might be analogous but inverse. Indeed, neither the lipid specificity nor the residues defining this specificity are conserved between epsin1 and Ent3p or Ent5p ENTH domains (Figure 1). The putative α0 helix is exacerbated in length in Ent3p and Ent5p when compared with other members of the epsin family, which could well account for a differing ability to deform membranes. To better understand the mechanism behind this process it will be of invaluable importance to obtain information on the structure of the ENTH domains of Ent3p and/or Ent5p.

Interestingly, the morphology of cells lacking Ent3p and Ent5p differs from both fab1Δ and vps27Δ cells, fab1Δ cells displaying one highly enlarged vacuole and the vps27Δ an enlarged endosomal/prevacuolar structure, whereas the ent3Δent5Δ cells showed fragmented and small vacuoles. Additionally, the vacuoles of Fab1p-depleted cells display a defect in acidification, whereas the fragmented vacuoles in ent3Δent5Δ cells are properly acidified. In fab1Δ cells the enlarged vacuole is the result of the lack of PtdIns(3,5)P2 synthesis (Bonangelino et al., 2002 blue right-pointing triangle), whereas it is likely that ent3Δent5Δ cells are not affected in PtdIns(3,5)P2 synthesis, but rather in PtdIns(3,5)P2 binding. The ent3Δent5Δ cells do not display classical class E phenotype, which might be due to their implication in different membrane trafficking pathways to the vacuole (Duncan et al., 2002 blue right-pointing triangle). Furthermore, Ent3p was recently shown to interact with Vti1p (Chidambaram et al., 2004 blue right-pointing triangle), a v-SNARE required for biosynthetic membrane trafficking pathways to the vacuole (Fischer von Mollard and Stevens, 1999 blue right-pointing triangle). Therefore absence of Ent3p and Ent5p could lead to a partial defect in the vesicle fusion with the vacuole, explaining the fragmented vacuolar phenotype and the CPY and ALP maturation delay that was observed in ent3-1, ent3-2 and ent3Δent5Δ mutant strains (Friant et al., 2003 blue right-pointing triangle).

The intracellular localization of Ent5p to the endosomes is affected in fab1Δ cells that are impaired in PtdIns(3,5)P2 synthesis, but not in vps27Δ cells. Furthermore, Vps27p is not required for the localization of Ent5p to the class E compartment of vps24Δ cells. These results show that the endosomal localization of Ent5p is mediated via its interaction with PtdIns(3,5)P2 and does not require Vps27p.

A picture, illustrating how sorting at the MVB could be regulated, is now emerging from our work and from previous reports. Vps27p, as a key player, binds to PtdIns(3)P rich domains on endosomal membranes, recruits ubiquitinated cargo proteins via its UIM and activates the ESCRT cascade (Bilodeau et al., 2003 blue right-pointing triangle; Katzmann et al., 2003 blue right-pointing triangle). These events might trigger an uncharacterized signaling event to Fab1p, which is localized to PtdIns(3)P-rich patches via its FYVE-domain and which, once activated, produces PtdIns(3,5)P2. Ent3p and Ent5p are then recruited to endosomal membranes on PtdIns(3,5)P2-rich patches and might induce membrane curvature and formation of MVB vesicles. By interacting with Vps27p-cargo complexes, Ent3p and Ent5p could ultimately contribute to the recruitment of these cargoes into nascent vesicles. This model for ubiquitinated cargo sorting into the MVB is similar to the one proposed for ubiquitin-dependent protein internalization in the endocytic pathway. The main difference is that the epsins required for endocytosis contain both the ENTH domain responsible for membrane deformations and the UIMs implicated in ubiquitinated receptor binding and in recruitment to the site of plasma membrane invaginations (Bilodeau et al., 2002 blue right-pointing triangle; Polo et al., 2002 blue right-pointing triangle; Raiborg et al., 2002 blue right-pointing triangle; Shih et al., 2002 blue right-pointing triangle). Here, in the case of the MVB pathway, a second effector, Vps27p, constitutes the link with ubiquinated cargo.

In summary, we identified Ent3p and Ent5p as the first connectors between the PtdIns(3,5)P2- and the Vps27p-ubiquitin-driven sorting machinery at the multivesicular body. Many aspects, such as the downstream effectors of Ent3p and Ent5p, other proteins found in complex with Vps27p, Ent3p, and Ent5p or the mechanism allowing recruitment of cargo into the vesicles remain to be elucidated. Genetic and molecular studies in yeast, structural studies as well as proteomics should provide better understanding of how Ent3p and Ent5p regulate ubiquitin-dependent sorting into the MVB.

Acknowledgments

We thank H.R. Pelham, S.D. Emr, B. Wendland, M.N. Hall, M.A. Lemmon, S.K. Lemmon, R.C. Piper, R. Duden, and J.M. Galan for sharing strains, plasmids and reagents, M. Bickle and R. Haguenauer-Tsapis for critical reading of the manuscript. This work was supported by a Fondation pour la Recherche Médicale (F.R.M.) and an Association Nationale pour la Recherche contre le SIDA (ANRS) fellowship to A.E. and by grants from the Association pour la Recherche sur le Cancer (to S.F.) and from the Programme Emergence 2002 (to E.I.P. and to F.L.).

Notes

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E03-11-0793. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E03-11-0793.

Abbreviations used: ALP, alkaline phosphatase; CPY, carboxypeptidase Y; ENTH, epsin NH2-terminal homology; ESCRT, endosomal sorting complex required for transport; FYVE domain, Fab1, YGL023, Vps27 and EEA1 domain; MVB, multivesicular body; PC, phosphatidylcholine; PtdIns(3,5)P2, phosphatidylinositol 3,5-bisphosphate; Ub, ubiquitin; UIM, Ub-interacting motif; Vps, vacuolar protein sorting.

References

  • Aguilar, R.C., Watson, H.A., and Wendland, B. (2003). The yeast epsin Ent1 is recruited to membranes through multiple independent interactions. J. Biol. Chem. 14, 14. [PubMed]
  • Babst, M., Katzmann, D.J., Estepa-Sabal, E.J., Meerloo, T., and Emr, S.D. (2002a). Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Dev. Cell 3, 271-282. [PubMed]
  • Babst, M., Katzmann, D.J., Snyder, W.B., Wendland, B., and Emr, S.D. (2002b). Endosome-associated complex, ESCRT-II, recruits transport machinery for protein sorting at the multivesicular body. Dev. Cell 3, 283-289. [PubMed]
  • Bilodeau, P.S., Urbanowski, J.L., Winistorfer, S.C., and Piper, R.C. (2002). The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting. Nat. Cell Biol. 4, 534-539. [PubMed]
  • Bilodeau, P.S., Winistorfer, S.C., Kearney, W.R., Robertson, A.D., and Piper, R.C. (2003). Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome. J. Cell Biol. 163, 237-243. [PMC free article] [PubMed]
  • Bonangelino, C.J., Catlett, N.L., and Weisman, L.S. (1997). Vac7p, a novel vacuolar protein, is required for normal vacuole inheritance and morphology. Mol. Cell. Biol. 17, 6847-6858. [PMC free article] [PubMed]
  • Bonangelino, C.J., Nau, J.J., Duex, J.E., Brinkman, M., Wurmser, A.E., Gary, J.D., Emr, S.D., and Weisman, L.S. (2002). Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p. J. Cell Biol. 156, 1015-1028. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Burda, P., Padilla, S.M., Sarkar, S., and Emr, S.D. (2002). Retromer function in endosome-to-Golgi retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase. J. Cell Sci. 115, 3889-3900. [PubMed]
  • Chidambaram, S., Mullers, N., Wiederhold, K., Haucke, V., and von Mollard, G.F. (2004). Specific interaction between SNAREs and Epsin N-terminal homology (ENTH) domains of Epsin-related proteins in trans-Golgi network to endosome transport. J. Biol. Chem. 279, 4175-4179. [PubMed]
  • Conibear, E., and Stevens, T.H. (1998). Multiple sorting pathways between the late Golgi and the vacuole in yeast. Biochim. Biophys. Acta 1404, 211-230. [PubMed]
  • Cosson, P., and Letourneur, F. (1994). Coatomer interaction with di-lysine endoplasmic reticulum retention motifs. Science 263, 1629-1631. [PubMed]
  • De Camilli, P., Chen, H., Hyman, J., Panepucci, E., Bateman, A., and Brunger, A.T. (2002). The ENTH domain. FEBS Lett. 513, 11-18. [PubMed]
  • Dove, S.K., McEwen, R.K., Mayes, A., Hughes, D.C., Beggs, J.D., and Michell, R.H. (2002). Vac14 controls PtdIns(3,5)P(2) synthesis and Fab1-dependent protein trafficking to the multivesicular body. Curr. Biol. 12, 885-893. [PubMed]
  • Duncan, M.C., Costaguta, G., and Payne, G.S. (2002). Yeast epsin-related proteins required for Golgi-endosome traffic define a gamma-adaptin ear-binding motif. Nat. Cell Biol. 16, 16. [PubMed]
  • Duncan, M.C., and Payne, G.S. (2003). ENTH/ANTH domains expand to the Golgi. Trends Cell Biol. 13, 211-215. [PubMed]
  • Epple, U.D., Eskelinen, E.-L., and Thumm, M. (2003). Intravacuolar membrane lysis in Saccharomyces cerevisiae. Does vacuolar targeting of Cvt17/Aut5p affect its function? J. Biol. Chem. 278, 7810-7821. [PubMed]
  • Felder, S., Miller, K., Moehren, G., Ullrich, A., Schlessinger, J., and Hopkins, C.R. (1990). Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body. Cell 61, 623-634. [PubMed]
  • Fischer von Mollard, G., and Stevens, T.H. (1999). The Saccharomyces cerevisiae v-SNARE Vti1p is required for multiple membrane transport pathways to the vacuole. Mol. Biol. Cell 10, 1719-1732. [PMC free article] [PubMed]
  • Ford, M.G., Mills, I.G., Peter, B.J., Vallis, Y., Praefcke, G.J., Evans, P.R., and McMahon, H.T. (2002). Curvature of clathrin-coated pits driven by epsin. Nature 419, 361-366. [PubMed]
  • Friant, S., Pecheur, E.I., Eugster, A., Michel, F., Lefkir, Y., Nourrisson, D., and Letourneur, F. (2003). Ent3p Is a PtdIns(3,5)P(2) effector required for protein sorting to the multivesicular body. Dev. Cell 5, 499-511. [PubMed]
  • Futter, C.E., Pearse, A., Hewlett, L.J., and 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. [PMC free article] [PubMed]
  • Gary, J.D., Wurmser, A.E., Bonangelino, C.J., Weisman, L.S., and Emr, S.D. (1998). Fab1p is essential for PtdIns(3)P 5-kinase activity and the maintenance of vacuolar size and membrane homeostasis. J. Cell Biol. 143, 65-79. [PMC free article] [PubMed]
  • Gaullier, J.M., Simonsen, A., D'Arrigo, A., Bremnes, B., Stenmark, H., and Aasland, R. (1998). FYVE fingers bind PtdIns(3)P. Nature 394, 432-433. [PubMed]
  • Gillooly, D.J., Morrow, I.C., Lindsay, M., Gould, R., Bryant, N.J., Gaullier, J.M., Parton, R.G., and Stenmark, H. (2000). Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells. EMBO J. 19, 4577-4588. [PMC free article] [PubMed]
  • Gouet, P., Courcelle, E., Stuart, D.I., and Metoz, F. (1999). ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305-308. [PubMed]
  • Hofmann, K., and Bucher, P. (1996). The UBA domain: a sequence motif present in multiple enzyme classes of the ubiquitination pathway. Trends Biochem. Sci. 21, 172-173. [PubMed]
  • Hofmann, K., and Falquet, L. (2001). A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems. Trends Biochem. Sci. 26, 347-350. [PubMed]
  • Holthuis, J.C.M., Nichols, B.J., Dhruvakumar, S., and Pelham, H.R.B. (1998). Two syntaxin homologues in the TGN/endosomal system of yeast. EMBO J. 17, 113-126. [PMC free article] [PubMed]
  • Katzmann, D.J., Babst, M., and 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. [PubMed]
  • Katzmann, D.J., Stefan, C.J., Babst, M., and Emr, S.D. (2003). Vps27 recruits ESCRT machinery to endosomes during MVB sorting. J. Cell Biol. 162, 413-423. [PMC free article] [PubMed]
  • Kay, B.K., Yamabhai, M., Wendland, B., and Emr, S.D. (1999). Identification of a novel domain shared by putative components of the endocytic and cytoskeletal machinery. Protein Sci. 8, 435-438. [PMC free article] [PubMed]
  • Marcusson, E.G., Horazdovsky, B.F., Cereghino, J.L., Gharakhanian, E., and Emr, S.D. (1994). The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene. Cell 77, 579-586. [PubMed]
  • Nelson, K.K., and Lemmon, S.K. (1993). Suppressors of clathrin deficiency: overexpression of ubiquitin rescues lethal strains of clathrin-deficient Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 521-532. [PMC free article] [PubMed]
  • Odorizzi, G., Babst, M., and Emr, S.D. (1998). Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body. Cell 95, 847-858. [PubMed]
  • Pelham, H. (2002). Insights from yeast endosomes. Curr. Opin. Cell Biol. 14, 454. [PubMed]
  • Piper, R.C., Bryant, N.J., and Stevens, T.H. (1997). The membrane protein alkaline phosphatase is delivered to the vacuole by a route that is distinct from the VPS-dependent pathway. J. Cell Biol. 138, 531-545. [PMC free article] [PubMed]
  • Polo, S., Sigismund, S., Faretta, M., Guidi, M., Capua, M.R., Bossi, G., Chen, H., De Camilli, P., and Di Fiore, P.P. (2002). A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins. Nature 416, 451-455. [PubMed]
  • Raiborg, C., Bache, K.G., Gillooly, D.J., Madshus, I.H., Stang, E., and Stenmark, H. (2002). Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes. Nat. Cell Biol. 4, 394-398. [PubMed]
  • Reggiori, F., and Pelham, H.R. (2001). Sorting of proteins into multivesicular bodies: ubiquitin-dependent and -independent targeting. EMBO J. 20, 5176-5186. [PMC free article] [PubMed]
  • Sankaran, V.G., Klein, D.E., Sachdeva, M.M., and Lemmon, M.A. (2001). High-affinity binding of a FYVE domain to phosphatidylinositol 3-phosphate requires intact phospholipid but not FYVE domain oligomerization. Biochemistry 40, 8581-8587. [PubMed]
  • Shaw, J.D., Hama, H., Sohrabi, F., DeWald, D.B., and Wendland, B. (2003). PtdIns(3,5)P2 is required for delivery of endocytic cargo into the multivesicular body. Traffic 4, 479-490. [PubMed]
  • Shih, S.C., Katzmann, D.J., Schnell, J.D., Sutanto, M., Emr, S.D., and Hicke, L. (2002). Epsins and Vps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis. Nat. Cell Biol. 4, 389-393. [PubMed]
  • Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680. [PMC free article] [PubMed]
  • Vida, T.A., and Emr, S.D. (1995). A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128, 779-792. [PMC free article] [PubMed]
  • Wendland, B., Steece, K.E., and Emr, S.D. (1999). Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis. EMBO J. 18, 4383-4393. [PMC free article] [PubMed]
  • Wishart, M.J., Taylor, G.S., and Dixon, J.E. (2001). Phoxy lipids: revealing PX domains as phosphoinositide binding modules. Cell 105, 817-820. [PubMed]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...