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Membrane transport: Ubiquitylation in endosomal sorting
R932
Dispatch
Membrane transport: Ubiquitylation in endosomal sorting Sophie Dupré, Christiane Volland and Rosine Haguenauer-Tsapis
In yeast, membrane proteins from the biosynthetic and endocytic pathways must be ubiquitylated for sorting to inward-budding vesicles in late endosomes, which give rise to multivesicular bodies. A conserved protein complex containing the yeast Vps23p or its mammalian counterpart Tsg101 may act as the ubiquitin receptor. Address: Institut Jacques Monod, Universités Paris VI and Paris VII, 2 place Jussieu, 75251 PARIS Cedex 05, France. E-mail: [email protected] Current Biology 2001, 11:R932–R934 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
At each stage in transport along the secretory and endocytic pathways protein sorting occurs. Some proteins remain within a given membrane, whereas others are selectively recruited to vesicles for transport to another organelle or membrane. This sorting involves short motifs within cargo protein sequences, and sometimes post-translational modifications. Recent studies have shown that ubiquitylation acts as an internalisation signal for plasma membrane proteins, particularly in the budding yeast Saccharomyces cerevisiae. We shall focus here on a newly defined role for ubiquitylation in the transport of membrane proteins from the trans-Golgi network to the lysosome/vacuole. Cargo membrane proteins are sorted, in a ubiquitin-dependent manner, to inwardbudding vesicles of late endosomes. This inward budding results in multivesicular body formation. Ubiquitylation — the covalent attachment of ubiquitin to specific lysine residues in target proteins — occurs in three steps, mediated sequentially by ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin–protein ligase (E32) enzymes. The last enzyme in the sequence, E3, is responsible for substrate recognition. Long multiubiquitin chains, in which the carboxy-terminal glycine of a ubiquitin molecule is linked to residue Lys48 of the preceding ubiquitin, target attached proteins to the proteasome for degradation. Ubiquitylation has been shown to have other functions, depending on alternative types of ubiquitin modification [1]. One of these other functions is a key role in endocytosis, and important new light has been shed on this process by several recent studies. In budding yeast, the downregulation of most plasma membrane receptor and transporter proteins requires their cell-surface ubiquitylation, followed by delivery to the lysosome/vacuole for degradation (Figure 1) [2,3]. The ubiquitylation of these proteins, often regulated by phosphorylation [3], is mediated by the ubiquitin–protein ligase
Rsp5p, and appears to involve addition of a single ubiquitin per target lysine — monoubiquitylation — or modification by Lys63-linked short chains. Nedd4, the human homologue of Rsp5p has been shown to be involved in the downregulation of several channel proteins [3]. Specific residues essential for internalisation have been identified on the surface of ubiquitin, and there is thought to be a ubiquitin receptor [4]. Interestingly, several proteins involved in the internalisation step of endocytosis [5,6] have the ubiquitin-interacting motifs UBA and/or UIM [7], but whether these proteins are true receptors has not been established. Ubiquitylated yeast plasma membrane proteins en route to the vacuole are sorted to early endosomes, and then to late endosomes, where they encounter proteins arriving from the trans-Golgi network. Membrane proteins from the biosynthetic and endocytic pathways are segregated in the late endosome for routing to the future vacuole lumen and membrane. The limiting membrane of the late endosome invaginates, budding into the lumen to form internal vesicles. These vesicles give rise to the so-called multivesicular bodies, which were first described in mammalian cells [8]. A subset of the membrane proteins, including cell surface proteins and biosynthetic proteins such as carboxypeptidase S, are sorted to invaginating vesicles, whereas others, such as dipeptidylaminopeptidase B (DPAP-B) remain in the limiting membrane of the endosome. The mature multivesicular body fuses with the vacuole, delivering the internal vesicles and their cargoes to the vacuole lumen. Endocytic proteins are then degraded by vacuolar proteases, and the vacuolar hydrolase carboxypeptidase S is cleaved from its transmembrane anchor to yield a mature soluble form. The proteins retained in the limiting membrane of the multivesicular body are delivered to the vacuolar membrane [9] (Figure 1). Until recently, little was known about the molecular mechanism underlying multivesicular body formation. Genetic studies in yeast led to the identification of a subset of vps (vacuolar protein sorting) mutants that accumulate an exaggerated late endosome, known as the ‘class E’ compartment. These mutants were found to be defective in multivesicular body formation [8]. One of the corresponding proteins is Vps23p, a homologue of the protein encoded by mammalian tumor susceptibility gene 101 (tsg101) [10]. Mutant vps23 cells are defective in carboxypeptidase S processing, and accumulate vacuolar and endocytic proteins in the class E compartment. Mammalian tsg101 mutant cells display recycling of the epidermal growth factor receptor after endocytosis, rather than normal lysosomal
Dispatch
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Figure 1 The role of ubiquitylation in protein sorting to multivesicular bodies. Plasma membrane receptors and transporters undergo cell surface Rsp5p-dependent ubiquitylation and are delivered to early endosomes (EE) and then to late endosomes (LE). Precursor carboxypeptidase S and transporters diverted from their normal route are ubiquitylated after exit from the Golgi compartment; DPAP-B escapes this post-translational modification. All these proteins are then routed to the LE. The ubiquitin tags are recognised by class E Vps complexes, which promote sorting to multivesicular body vesicles, whereas nonubiquitylated DPAP-B remains in the noninvaginated LE membrane. Doa4p removes the ubiquitin residues from target proteins before these proteins enter the multivesicular body, thereby recycling ubiquitin to the cytoplasm. Fusion of the multivesicular body with the vacuole results in the delivery of DPAP-B to the vacuolar membrane, and of multivesicular body vesicles to the lumen of the vacuole, where vacuolar proteases process carboxypeptidase S and degrade receptors and transporters. Ubiquitin modification is represented by a unique symbol, whether it corresponds to monoubiquitylation, as for a receptor [2] or modification by a small number of residues, as described for carboxypeptidases [9] or plasma membrane transporters [3].
Plasma membrane
Cytosol
TGN
EE Doa4p Vesicle
Class E Vps
LE Vacuole Current Biology
Plasma membrane receptor or transporter Transporter diverted from its normal route
sorting [10]. Vps23p and Tsg101 have a ubiquitin-conjugating (UBC)-like domain lacking the catalytic cysteine. A point mutation of a conserved residue in this domain abolishes VPS23 function [9]. It has therefore been suggested that either the multivesicular body sorting machinery is regulated by ubiquitylation, or Vps23p binds to ubiquitylated membrane proteins to direct them into multivesicular bodies [8]. In agreement with this idea, several proteins coming from the biosynthetic pathway or the plasma membrane are indeed missorted to the vacuolar membrane instead of the vacuolar lumen in mutant cells with low ubiquitin levels [11], and correct sorting is restored by ubiquitin overproduction [12,13]. Elegant recent studies in yeast, by Katzman et al [9], Reggiori and Pelham [13] and Urbanowski and Piper [14], have now established that the ubiquitylation of cargo yeast proteins plays a key role in their internalisation into the multivesicular body. Two biosynthetic membrane proteins destined for the vacuolar lumen, carboxypeptidase S and the polyphosphatase Phm5p, are both ubiquitylated at one target lysine, with the addition of a limited number of ubiquitin moieties. Mutation of the target lysine in these proteins was found to result in their missorting and accumulation in the vacuolar membrane. Normal sorting to
Precursor carboxypeptidase
Ubiquitin
Mature carboxypeptidase
DPAP-B
multivesicular body vesicles was shown to be rescued by the fusion of a single ubiquitin molecule at the amino terminus of the mutant proteins [9,13]. The carboxypeptidase S sequence including the target lysine, or ubiquitin itself, has been shown to act as a signal which directs into the vacuolar lumen resident vacuolar membrane-bound proteins, such as DPAP-B [9], the iron transporter Fth1p and even Vps10p, a protein that normally recycles to the trans-Golgi network [14]. Before invagination, the ubiquitin isopeptidase, Doa4p which accumulates in the class E compartment in vps mutants [11], removes ubiquitin from biosynthetic cargo proteins and from plasma membrane proteins that have undergone endocytosis [9,15]. A lack of deubiquitylation does not, however, prevent sorting into invaginated vesicles [15]. It should be noted that ubiquitylation may not be the unique sorting signal, as at least one membrane protein, Sna3p, is targeted to the vacuolar lumen without undergoing direct ubiquitylation [13]. Important new insight into the function of Vps23p in protein sorting to the internal vesicles of the multivesicular body has been provided by Katzman et al [9]. Vps23p is part of a 350 kDa protein complex, ESCRT-I — for endosomal complex required for transport — which also contains multiple copies of two other class E proteins, Vps28p
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and Vps37p. When part of the ESCRT-I complex, Vps23p interacts in vivo with ubiquitylated carboxypeptidase S. The UBC-like domain of Vps23p is required for this interaction. ESCRT-I is thus a conserved component of the endosomal sorting machinery, which recognises ubiquitylated cargo proteins at the endosome. Other ESCRT complexes probably act further downstream to regulate protein sorting in the multivesicular body pathway [9]. The ubiquitin–protein ligase responsible for the ubiquitylation of carboxypeptidase S and Phm5p has not yet been identified, but several groups working on yeast plasma membrane transporters have provided clues to the identity of this E3 enzyme. In response to changes in nutrient status, some permeases — Tat2p and Gap1p — are sorted directly from the Golgi apparatus to the vacuole, instead of undergoing normal plasma membrane targeting. This unusual sorting requires Rsp5p-dependent ubiquitylation [16–18] and in rsp5 mutant cells, Tat2p has been shown to be missorted to the vacuolar membrane instead of the vacuolar lumen [16]. The critical Rsp5p-dependent step in the trans-Golgi network-to-vacuole transport of Gap1p appears to take place after exit from the Golgi compartment, but before the step involving the endosomal t-SNARE Pep12p [18], just as reported for carboxypeptidase S ubiquitylation [9]. The similarity in fate of these diverted transporters, carboxypeptidase S and Phm5p suggests that Rsp5p may be the ubiquitin protein ligase involved in the ubiquitylation of all proteins targeted to the multivesicular body pathway. The demonstration that ubiquitin acts as a signal for sorting to multivesicular bodies highlights the reported role of ubiquitylation in late stages of the endocytic pathway in mammalian cells [19,20]. Somewhat similar to the sorting role of ubiquitylation at two steps of intracellular transport is its occurrence in virus particle release from infected cells. The viral Gag polyprotein, which forms the structural component of the budding machinery, requires ubiquitylation to effect budding. Strikingly, the Gag protein of Rous sarcoma virus was recently reported to interact with the Nedd4 ubiquitin–protein ligase [21], and the Gag protein of HIV-1 was found to interact with the UBC-like domain of Tsg101 [22]. Deciphering the roles of ubiquitylation in both internalisation at the plasma membrane and invagination into multivesicular bodies should also provide insight into the enigmatic role of ubiquitin in the budding of enveloped viruses from the plasma membrane. References 1. Weissman AM: Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol 2001, 2:169-178. 2. Hicke L: Gettin’ down with ubiquitin: turning off cell-surface receptors transporters and channels. Trends Cell Biol 1999, 9:107-112.
3. Rotin D, Staub O, Haguenauer-Tsapis R: Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases. J Membr Biol 2000, 176:1-17. 4. Shih SC, Sloper-Mould KE, Hicke L: Monoubiquitin carries a novel internalization signal that is appended to active receptors. EMBO J 2000, 17:187-198. 5. Gagny B, Wiederkehr A, Dumoulin P, Winsor B, Riezman H, Haguenauer-Tsapis R: A novel EH domain protein of Saccharomyces cerevisiae, Ede1p, involved in endocytosis. J Cell Sci 2000, 113:3309-3319. 6. Wendland B, Steece K. E, Emr SD: Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis. EMBO J 1999, 18:4383-4393. 7. Hofmann K, Falquet L: A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems. Trends Biochem Sci 2001, 26:347-350. 8. Lemmon SK, Traub, LM: Sorting in the endosomal system in yeast and animal cells. Curr Opin Cell Biol 2000, 12:457-466. 9. Katzmann DJ, Babst M, Emr SD: Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 2001, 106:145-155. 10. Babst M, Odorizzi G, Estepa EJ, Emr SD: Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking. Traffic 2000, 1:248-258. 11. Amerik AY, Nowak J, Swaminathan S, Hochstrasser M: The doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways. Mol Biol Cell 2000, 11:3365-3380. 12. Losko S, Kopp F, Kranz A, Kolling R: Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 Mutant. Mol Biol Cell 2001, 12:1047-1059. 13. Reggiori F, Pelham HRB: Sorting of proteins into mutivesicular bodies: ubiquitin dependent and independent targeting. EMBO J 2001, 20:5176-5186. 14. Urbanowski J, Piper RC: Ubiquitin sorts proteins into the lumenal degradative compartment of the late endosome/vacuole. Traffic 2001, 20:622-630. 15. Dupre S, Haguenauer-Tsapis R: Deubiquitination step in the endocytic pathway of yeast plasma membrane proteins: crucial role of Doa4p ubiquitin isopeptidase. Mol Cell Biol 2001, 21:4482-4494. 16. Beck T, Schmidt A, Hall MN: Starvation induces vacuolar targeting and degradation of the tryptophan permease in yeast. J Cell Biol 1999, 146:1227-1238. 17. Helliwell SB, Losko S, Kaiser CA: Components of a ubiquitin ligase complex specify polyubiquitination and intracellular trafficking of the general amino acid permease. J Cell Biol 2001, 153:649-662. 18. Soetens O, De Craene JO, Andre B: Ubiquitin is required for sorting to the vacuole of the yeast Gap1 permease. J Biol Chem 2001, 10:10. 19. Strous GJ, Govers R: The ubiquitin-proteasome system and endocytosis. J Cell Sci 1999, 112:1417-1423. 20. Rocca A, Lamaze C, Subtil A, Dautry-Varsat A: Involvement of the ubiquitin/proteasome pathway in sorting of the interleukin 2 receptor β chain to late endocytic compartments. Mol Biol Cell 2001, 12:1294-1301. 21. Patnaik A, Chau V, Wills JW: Ubiquitin is part of the retrovirus budding machinery. Proc Natl Acad Sci USA 2000, 97:1306913074. 22. VerPlank L, Bouamr F, LaGrassa TJ, Agresta B, Kikonyogo A, Leis J, Carter CA: Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc Natl Acad Sci USA 2001, 98:7724-7729.
Dispatch
Membrane transport: Ubiquitylation in endosomal sorting Sophie Dupré, Christiane Volland and Rosine Haguenauer-Tsapis
In yeast, membrane proteins from the biosynthetic and endocytic pathways must be ubiquitylated for sorting to inward-budding vesicles in late endosomes, which give rise to multivesicular bodies. A conserved protein complex containing the yeast Vps23p or its mammalian counterpart Tsg101 may act as the ubiquitin receptor. Address: Institut Jacques Monod, Universités Paris VI and Paris VII, 2 place Jussieu, 75251 PARIS Cedex 05, France. E-mail: [email protected] Current Biology 2001, 11:R932–R934 0960-9822/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
At each stage in transport along the secretory and endocytic pathways protein sorting occurs. Some proteins remain within a given membrane, whereas others are selectively recruited to vesicles for transport to another organelle or membrane. This sorting involves short motifs within cargo protein sequences, and sometimes post-translational modifications. Recent studies have shown that ubiquitylation acts as an internalisation signal for plasma membrane proteins, particularly in the budding yeast Saccharomyces cerevisiae. We shall focus here on a newly defined role for ubiquitylation in the transport of membrane proteins from the trans-Golgi network to the lysosome/vacuole. Cargo membrane proteins are sorted, in a ubiquitin-dependent manner, to inwardbudding vesicles of late endosomes. This inward budding results in multivesicular body formation. Ubiquitylation — the covalent attachment of ubiquitin to specific lysine residues in target proteins — occurs in three steps, mediated sequentially by ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin–protein ligase (E32) enzymes. The last enzyme in the sequence, E3, is responsible for substrate recognition. Long multiubiquitin chains, in which the carboxy-terminal glycine of a ubiquitin molecule is linked to residue Lys48 of the preceding ubiquitin, target attached proteins to the proteasome for degradation. Ubiquitylation has been shown to have other functions, depending on alternative types of ubiquitin modification [1]. One of these other functions is a key role in endocytosis, and important new light has been shed on this process by several recent studies. In budding yeast, the downregulation of most plasma membrane receptor and transporter proteins requires their cell-surface ubiquitylation, followed by delivery to the lysosome/vacuole for degradation (Figure 1) [2,3]. The ubiquitylation of these proteins, often regulated by phosphorylation [3], is mediated by the ubiquitin–protein ligase
Rsp5p, and appears to involve addition of a single ubiquitin per target lysine — monoubiquitylation — or modification by Lys63-linked short chains. Nedd4, the human homologue of Rsp5p has been shown to be involved in the downregulation of several channel proteins [3]. Specific residues essential for internalisation have been identified on the surface of ubiquitin, and there is thought to be a ubiquitin receptor [4]. Interestingly, several proteins involved in the internalisation step of endocytosis [5,6] have the ubiquitin-interacting motifs UBA and/or UIM [7], but whether these proteins are true receptors has not been established. Ubiquitylated yeast plasma membrane proteins en route to the vacuole are sorted to early endosomes, and then to late endosomes, where they encounter proteins arriving from the trans-Golgi network. Membrane proteins from the biosynthetic and endocytic pathways are segregated in the late endosome for routing to the future vacuole lumen and membrane. The limiting membrane of the late endosome invaginates, budding into the lumen to form internal vesicles. These vesicles give rise to the so-called multivesicular bodies, which were first described in mammalian cells [8]. A subset of the membrane proteins, including cell surface proteins and biosynthetic proteins such as carboxypeptidase S, are sorted to invaginating vesicles, whereas others, such as dipeptidylaminopeptidase B (DPAP-B) remain in the limiting membrane of the endosome. The mature multivesicular body fuses with the vacuole, delivering the internal vesicles and their cargoes to the vacuole lumen. Endocytic proteins are then degraded by vacuolar proteases, and the vacuolar hydrolase carboxypeptidase S is cleaved from its transmembrane anchor to yield a mature soluble form. The proteins retained in the limiting membrane of the multivesicular body are delivered to the vacuolar membrane [9] (Figure 1). Until recently, little was known about the molecular mechanism underlying multivesicular body formation. Genetic studies in yeast led to the identification of a subset of vps (vacuolar protein sorting) mutants that accumulate an exaggerated late endosome, known as the ‘class E’ compartment. These mutants were found to be defective in multivesicular body formation [8]. One of the corresponding proteins is Vps23p, a homologue of the protein encoded by mammalian tumor susceptibility gene 101 (tsg101) [10]. Mutant vps23 cells are defective in carboxypeptidase S processing, and accumulate vacuolar and endocytic proteins in the class E compartment. Mammalian tsg101 mutant cells display recycling of the epidermal growth factor receptor after endocytosis, rather than normal lysosomal
Dispatch
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Figure 1 The role of ubiquitylation in protein sorting to multivesicular bodies. Plasma membrane receptors and transporters undergo cell surface Rsp5p-dependent ubiquitylation and are delivered to early endosomes (EE) and then to late endosomes (LE). Precursor carboxypeptidase S and transporters diverted from their normal route are ubiquitylated after exit from the Golgi compartment; DPAP-B escapes this post-translational modification. All these proteins are then routed to the LE. The ubiquitin tags are recognised by class E Vps complexes, which promote sorting to multivesicular body vesicles, whereas nonubiquitylated DPAP-B remains in the noninvaginated LE membrane. Doa4p removes the ubiquitin residues from target proteins before these proteins enter the multivesicular body, thereby recycling ubiquitin to the cytoplasm. Fusion of the multivesicular body with the vacuole results in the delivery of DPAP-B to the vacuolar membrane, and of multivesicular body vesicles to the lumen of the vacuole, where vacuolar proteases process carboxypeptidase S and degrade receptors and transporters. Ubiquitin modification is represented by a unique symbol, whether it corresponds to monoubiquitylation, as for a receptor [2] or modification by a small number of residues, as described for carboxypeptidases [9] or plasma membrane transporters [3].
Plasma membrane
Cytosol
TGN
EE Doa4p Vesicle
Class E Vps
LE Vacuole Current Biology
Plasma membrane receptor or transporter Transporter diverted from its normal route
sorting [10]. Vps23p and Tsg101 have a ubiquitin-conjugating (UBC)-like domain lacking the catalytic cysteine. A point mutation of a conserved residue in this domain abolishes VPS23 function [9]. It has therefore been suggested that either the multivesicular body sorting machinery is regulated by ubiquitylation, or Vps23p binds to ubiquitylated membrane proteins to direct them into multivesicular bodies [8]. In agreement with this idea, several proteins coming from the biosynthetic pathway or the plasma membrane are indeed missorted to the vacuolar membrane instead of the vacuolar lumen in mutant cells with low ubiquitin levels [11], and correct sorting is restored by ubiquitin overproduction [12,13]. Elegant recent studies in yeast, by Katzman et al [9], Reggiori and Pelham [13] and Urbanowski and Piper [14], have now established that the ubiquitylation of cargo yeast proteins plays a key role in their internalisation into the multivesicular body. Two biosynthetic membrane proteins destined for the vacuolar lumen, carboxypeptidase S and the polyphosphatase Phm5p, are both ubiquitylated at one target lysine, with the addition of a limited number of ubiquitin moieties. Mutation of the target lysine in these proteins was found to result in their missorting and accumulation in the vacuolar membrane. Normal sorting to
Precursor carboxypeptidase
Ubiquitin
Mature carboxypeptidase
DPAP-B
multivesicular body vesicles was shown to be rescued by the fusion of a single ubiquitin molecule at the amino terminus of the mutant proteins [9,13]. The carboxypeptidase S sequence including the target lysine, or ubiquitin itself, has been shown to act as a signal which directs into the vacuolar lumen resident vacuolar membrane-bound proteins, such as DPAP-B [9], the iron transporter Fth1p and even Vps10p, a protein that normally recycles to the trans-Golgi network [14]. Before invagination, the ubiquitin isopeptidase, Doa4p which accumulates in the class E compartment in vps mutants [11], removes ubiquitin from biosynthetic cargo proteins and from plasma membrane proteins that have undergone endocytosis [9,15]. A lack of deubiquitylation does not, however, prevent sorting into invaginated vesicles [15]. It should be noted that ubiquitylation may not be the unique sorting signal, as at least one membrane protein, Sna3p, is targeted to the vacuolar lumen without undergoing direct ubiquitylation [13]. Important new insight into the function of Vps23p in protein sorting to the internal vesicles of the multivesicular body has been provided by Katzman et al [9]. Vps23p is part of a 350 kDa protein complex, ESCRT-I — for endosomal complex required for transport — which also contains multiple copies of two other class E proteins, Vps28p
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and Vps37p. When part of the ESCRT-I complex, Vps23p interacts in vivo with ubiquitylated carboxypeptidase S. The UBC-like domain of Vps23p is required for this interaction. ESCRT-I is thus a conserved component of the endosomal sorting machinery, which recognises ubiquitylated cargo proteins at the endosome. Other ESCRT complexes probably act further downstream to regulate protein sorting in the multivesicular body pathway [9]. The ubiquitin–protein ligase responsible for the ubiquitylation of carboxypeptidase S and Phm5p has not yet been identified, but several groups working on yeast plasma membrane transporters have provided clues to the identity of this E3 enzyme. In response to changes in nutrient status, some permeases — Tat2p and Gap1p — are sorted directly from the Golgi apparatus to the vacuole, instead of undergoing normal plasma membrane targeting. This unusual sorting requires Rsp5p-dependent ubiquitylation [16–18] and in rsp5 mutant cells, Tat2p has been shown to be missorted to the vacuolar membrane instead of the vacuolar lumen [16]. The critical Rsp5p-dependent step in the trans-Golgi network-to-vacuole transport of Gap1p appears to take place after exit from the Golgi compartment, but before the step involving the endosomal t-SNARE Pep12p [18], just as reported for carboxypeptidase S ubiquitylation [9]. The similarity in fate of these diverted transporters, carboxypeptidase S and Phm5p suggests that Rsp5p may be the ubiquitin protein ligase involved in the ubiquitylation of all proteins targeted to the multivesicular body pathway. The demonstration that ubiquitin acts as a signal for sorting to multivesicular bodies highlights the reported role of ubiquitylation in late stages of the endocytic pathway in mammalian cells [19,20]. Somewhat similar to the sorting role of ubiquitylation at two steps of intracellular transport is its occurrence in virus particle release from infected cells. The viral Gag polyprotein, which forms the structural component of the budding machinery, requires ubiquitylation to effect budding. Strikingly, the Gag protein of Rous sarcoma virus was recently reported to interact with the Nedd4 ubiquitin–protein ligase [21], and the Gag protein of HIV-1 was found to interact with the UBC-like domain of Tsg101 [22]. Deciphering the roles of ubiquitylation in both internalisation at the plasma membrane and invagination into multivesicular bodies should also provide insight into the enigmatic role of ubiquitin in the budding of enveloped viruses from the plasma membrane. References 1. Weissman AM: Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol 2001, 2:169-178. 2. Hicke L: Gettin’ down with ubiquitin: turning off cell-surface receptors transporters and channels. Trends Cell Biol 1999, 9:107-112.
3. Rotin D, Staub O, Haguenauer-Tsapis R: Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases. J Membr Biol 2000, 176:1-17. 4. Shih SC, Sloper-Mould KE, Hicke L: Monoubiquitin carries a novel internalization signal that is appended to active receptors. EMBO J 2000, 17:187-198. 5. Gagny B, Wiederkehr A, Dumoulin P, Winsor B, Riezman H, Haguenauer-Tsapis R: A novel EH domain protein of Saccharomyces cerevisiae, Ede1p, involved in endocytosis. J Cell Sci 2000, 113:3309-3319. 6. Wendland B, Steece K. E, Emr SD: Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis. EMBO J 1999, 18:4383-4393. 7. Hofmann K, Falquet L: A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems. Trends Biochem Sci 2001, 26:347-350. 8. Lemmon SK, Traub, LM: Sorting in the endosomal system in yeast and animal cells. Curr Opin Cell Biol 2000, 12:457-466. 9. Katzmann DJ, Babst M, Emr SD: Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 2001, 106:145-155. 10. Babst M, Odorizzi G, Estepa EJ, Emr SD: Mammalian tumor susceptibility gene 101 (TSG101) and the yeast homologue, Vps23p, both function in late endosomal trafficking. Traffic 2000, 1:248-258. 11. Amerik AY, Nowak J, Swaminathan S, Hochstrasser M: The doa4 deubiquitinating enzyme is functionally linked to the vacuolar protein-sorting and endocytic pathways. Mol Biol Cell 2000, 11:3365-3380. 12. Losko S, Kopp F, Kranz A, Kolling R: Uptake of the ATP-binding cassette (ABC) transporter Ste6 into the yeast vacuole is blocked in the doa4 Mutant. Mol Biol Cell 2001, 12:1047-1059. 13. Reggiori F, Pelham HRB: Sorting of proteins into mutivesicular bodies: ubiquitin dependent and independent targeting. EMBO J 2001, 20:5176-5186. 14. Urbanowski J, Piper RC: Ubiquitin sorts proteins into the lumenal degradative compartment of the late endosome/vacuole. Traffic 2001, 20:622-630. 15. Dupre S, Haguenauer-Tsapis R: Deubiquitination step in the endocytic pathway of yeast plasma membrane proteins: crucial role of Doa4p ubiquitin isopeptidase. Mol Cell Biol 2001, 21:4482-4494. 16. Beck T, Schmidt A, Hall MN: Starvation induces vacuolar targeting and degradation of the tryptophan permease in yeast. J Cell Biol 1999, 146:1227-1238. 17. Helliwell SB, Losko S, Kaiser CA: Components of a ubiquitin ligase complex specify polyubiquitination and intracellular trafficking of the general amino acid permease. J Cell Biol 2001, 153:649-662. 18. Soetens O, De Craene JO, Andre B: Ubiquitin is required for sorting to the vacuole of the yeast Gap1 permease. J Biol Chem 2001, 10:10. 19. Strous GJ, Govers R: The ubiquitin-proteasome system and endocytosis. J Cell Sci 1999, 112:1417-1423. 20. Rocca A, Lamaze C, Subtil A, Dautry-Varsat A: Involvement of the ubiquitin/proteasome pathway in sorting of the interleukin 2 receptor β chain to late endocytic compartments. Mol Biol Cell 2001, 12:1294-1301. 21. Patnaik A, Chau V, Wills JW: Ubiquitin is part of the retrovirus budding machinery. Proc Natl Acad Sci USA 2000, 97:1306913074. 22. VerPlank L, Bouamr F, LaGrassa TJ, Agresta B, Kikonyogo A, Leis J, Carter CA: Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag). Proc Natl Acad Sci USA 2001, 98:7724-7729.