Third Intracellular Proteolysis Meeting

Versatile role of the yeast ubiquitin ligase Rsp5p in intracellular trafficking

Naima Belgareh-Touzé, Sébastien Léon, Zoi Erpapazoglou, Marta Stawiecka-Mirota, Danièle Urban-Grimal, Rosine Haguenauer-Tsapis

Abstract

The ubiquitin ligase (E3) Rsp5p is the only member of the Nedd (neural-precursor-cell-expressed, developmentally down-regulated) 4 family of E3s present in yeast. Rsp5p has several proteasome-independent functions in membrane protein trafficking, including a role in the ubiquitination of most plasma membrane proteins, leading to their endocytosis. Rsp5p is also required for the ubiquitination of endosomal proteins, leading to their sorting to the internal vesicles of MVBs (multivesicular bodies). Rsp5p catalyses the attachment of non-conventional ubiquitin chains, linked through ubiquitin Lys-63, to some endocytic and MVB cargoes. This modification appears to be required for efficient sorting, possibly because these chains have a greater affinity for the ubiquitin-binding domains present within endocytic or MVB sorting complexes. The mechanisms involved in the recognition of plasma membrane and MVB substrates by Rsp5p remain unclear. A subset of Rsp5/Nedd4 substrates have a ‘PY motif’ and are recognized directly by the WW (Trp-Trp) domains of Rsp5p. Most Rsp5p substrates do not carry PY motifs, but some may depend on PY-containing proteins for their ubiquitination by Rsp5p, consistent with the latter's acting as specificity factors or adaptors. As in other ubiquitin-conjugating systems, these adaptors are also Rsp5p substrates and undergo ubiquitin-dependent trafficking. In the present review, we discuss recent examples illustrating the role of Rsp5p in membrane protein trafficking and providing new insights into the regulation of this E3 by adaptor proteins.

  • E3 adaptor
  • endocytosis
  • multivesicular body (MVB)
  • Rsp5p
  • membrane protein trafficking
  • yeast

Introduction

Ubiquitination was initially described as promoting proteasomal degradation, and has since been shown to regulate other processes, including DNA repair, signalling and trafficking. In the present review, we will focus on the role of the yeast ubiquitin protein ligase Rsp5p in several steps of intracellular trafficking.

Protein ubiquitination is a reversible post-translational modification in which the 76-amino-acid peptide ubiquitin is covalently linked, via its C-terminal glycine residue, to the ϵ-amino group of the lysine residues of target proteins. This process involves sequential {E1 (ubiquitin-activating), E2 [UBC (ubiquitin-conjugating)] and E3 (ubiquitin-protein ligase)} enzyme activities, with the E3 responsible for target recognition [1]. E3 enzymes form several families, the two main families being RING (really interesting new gene)-finger and HECT (homologous to E6-associated protein C-terminus) E3 proteins. RING-finger E3s act as scaffolds, bringing E2 and the substrate into contact. HECT-domain E3s are directly involved in catalysis: the activated ubiquitin is transferred from the E2 to an internal cysteine residue on the E3 and is then conjugated to a lysine residue within the target. As ubiquitin itself carries several lysine residues (Lys-6, -11, -27, -29, -33, -48 and -63), multi-ubiquitin chains are frequently formed. Lys-48-linked multi-ubiquitin chains, at least four ubiquitin units long, are potent signals leading to recognition and degradation by the 26S proteasome. Lys-63-linked multi-ubiquitin chains are involved in other functions, including DNA repair, the activation of specific kinases and endocytosis. Finally, ubiquitination is reversible, and ubiquitin can be removed by DUB enzymes (deubiquitination enzymes) [1].

HECT-containing E3s include the family of Nedd (neural-precursor-cell-expressed, developmentally down-regulated) 4/Nedd4-like ubiquitin ligases, of which there are nine in humans and one in Saccharomyces cerevisiae: the essential protein Rsp5p. These proteins have a modular structure: an N-terminal C2 domain (mainly known as a lipid-binding domain), two to four WW (Trp-Trp) domains (protein–protein interaction modules that bind a short recognition motif called a PY motif: [L/P]PxY) and a catalytic C-terminal HECT domain. Rsp5p has multiple functions, including roles in mRNA export [2] or the processing of transcription factors [3]. Its function in trafficking has already been documented (reviewed in [47]). Here, we summarize recent data and discuss some of the questions that have been raised concerning the mode of action of Rsp5p in trafficking.

Role of Rsp5p in the internalization step of endocytosis

The role of Rsp5p in trafficking was initially discovered in a genetic screen investigating the ammonium-induced down-regulation (‘inactivation’) of yeast amino-acid transporters. Several npi (nitrogen permease inactivator) mutants had been isolated in which the general amino-acid permease Gap1p was not inactivated by ammonium. Cloning and sequencing of the NPI1 gene showed that it encoded the ubiquitin ligase Rsp5p. Both Gap1p and another transporter, the Fur4p uracil permease, were then shown to undergo Rsp5p-dependent ubiquitination at the plasma membrane, a modification required for their internalization and subsequent proteasome-independent vacuolar degradation. Mutation of the target lysine residues in these transporters strongly impaired internalization of these proteins (reviewed in [4,8]). Rsp5p-mediated ubiquitination and internalization appear to be a general feature of most plasma membrane proteins in yeast [4,9] (Figure 1). Ubiquitin-dependent endocytosis also occurs in mammalian cells, mediated either by Nedd4/Nedd4-like E3s or by RING-finger E3s, but this process is less general in mammals than in yeast [4,6,7,9].

Figure 1 Multiple roles of Rsp5p in protein trafficking

A multispanning transporter targeted to the plasma membrane (Pl Mb) undergoes phosphorylation (results not shown) followed by Rsp5p-dependent ubiquitination (UbK63-linked oligo-ubiquitination), triggering its internalization. The same transporter can be directly routed to the endosomal pathway, therefore following the Vps pathway. This pathway can be used by other proteins, such as a membrane PY-motif (black rectangle)-containing protein. Both proteins undergo Rsp5p-dependent ubiquitination in their sorting from the Golgi apparatus to endosomes. Whereas the transporter displays adaptor-dependent ubiquitination at MVB, the PY-motif-containing protein interacts directly with Rsp5p, leading to its ubiquitination (UbK63-linked chains). Ubiquitinated proteins originating from the Golgi or the plasma membrane undergo deubiquitination prior to their sorting to MVB internal vesicles. After fusion of MVBs with the vacuole, these proteins are degraded by vacuolar proteases.

The observation of Rsp5p-mediated endocytosis in yeast constituted a major step towards deciphering the mechanisms involved in the internalization step of endocytosis in yeast, but it also raised new questions, concerning substrate recognition in particular. The epithelial sodium channel ENaC, one of the first plasma membrane substrates of Nedd4 to be described in mammals, carries several PY motifs that are recognized by Nedd4 through a PY–WW interaction [10]. This mode of recognition has often been considered to be general, and the WW domains of Rsp5/Nedd4 E3s are often presented as substrate recognition modules. Indeed, some Rsp5p substrates carry PY motifs, and display PY–WW-based interaction with Rsp5p [11], but this is not the case for plasma membrane endocytic substrates [4]. It has been suggested that PY-containing proteins may act as Rsp5p-substrate adaptors. The only PY-containing proteins characterized so far that interact with Rsp5p and regulate the endocytosis of a subset of plasma membrane proteins are the homologous, cytosolic proteins Bul1/2. These proteins are required for the Rsp5p-mediated ubiquitination and internalization of several endocytic substrates [1214]. However, the role of these proteins, which have also been presented as E4 enzymes (involved in the extension of ubiquitin chains) [15], remains unclear. The mechanism underlying Bul1/2 function remains to be defined, and other potential adaptors at the plasma membrane remain to be identified. There is also an additional level of complexity concerning the recognition of Rsp5p plasma membrane substrates. In addition to basal endocytosis, most yeast plasma membrane proteins display regulated ubiquitination and internalization. For example, the ubiquitination and internalization of Gap1p, Tat2p and Ctr1p are triggered by ammonium, high tryptophan, and high copper concentrations respectively [8]. It is tempting to hypothesize that phosphorylation events may play a role in substrate recognition: indeed, the phosphorylation of Ste2p (triggered by its ligand, α-factor) and Fur4p is required for the ubiquitination of these proteins [4,9]. This post-translational modification may result in conformational changes, rendering certain lysine residues accessible for ubiquitination. It remains unclear whether such conformational changes may trigger the direct interaction of Rsp5p with substrates or with potential adaptors.

The first observations of ubiquitin-dependent internalization rapidly led to questions concerning the mechanism by which these substrates escaped recognition by the proteasome. The use of ubiquitin point mutants (mutation of lysine into arginine) showed that Fur4p and Gap1p were modified by short ubiquitin chains (two to three ubiquitin moieties long, oligo-ubiquitination) linked via their Lys-63 residues [16,17], and that this modification was required for efficient endocytosis. By contrast, the α-factor receptor Ste2p has been reported to undergo primarily mono-ubiquitination [18]. Two other yeast transporters were recently shown to be modified by Rsp5p, with the attachment of UbK63 (ubiquitin Lys-63)-linked ubiquitin chains [19,19a]. These observations are consistent with recent studies showing that Rsp5p preferentially assembles this type of ubiquitin chain in vitro and in vivo [20]. A number of plasma membrane transporters or cell surface proteins also undergo modification by Lys-63-linked ubiquitin chains in mammalian cells [6,2123]. This type of ubiquitination is carried out by Nedd4 in the case of the dopamine DAT transporter [6].

The role of Rsp5p in sorting to MVBs (multivesicular bodies)

In addition to its role as an internalization signal at the plasma membrane, ubiquitination is also required for the sortting of membrane proteins originating from the Golgi apparatus or the plasma membrane to the internal vesicles of MVBs [24]. Yeast MVB cargoes have been shown to undergo ubiquitination. Inhibition of this ubiquitination abolishes MVB sorting, resulting in the recovery of cargoes at the vacuolar membrane rather than in the vacuolar lumen, after the fusion of MVBs with the vacuole (reviewed in [24]). Genetic analyses in yeast have led to the discovery of four protein complexes, the ESCRTs (endosomal sorting complexes required for transport) (ESCRT-0, -I, -II and -III), conserved from yeast to humans and involved in the recognition of ubiquitinated cargoes and their sorting to the internal vesicles of MVBs. ESCRT-0, -I and -II each carry at least one subunit bearing a ubiquitin-binding domain [24]. Extensive characterization of the ESCRT complexes preceded identification of the E3 involved in the ubiquitination of MVB cargoes, which turned out to be Rsp5p [2527]. The MVB substrates of Rsp5p identified to date include two vacuolar enzymes, namely the carboxypeptidase S1, Cps1p, and the polyphosphate phosphatase Phm5p, Sna3p (a membrane protein of unknown function) and several transporters directly sorted from the Golgi apparatus to the vacuole, bypassing plasma membrane delivery, under certain nutrient/substrate conditions [8,11,24,28,29]. Nedd4 and the Nedd4-like E3 AIP4/Itch have also been shown to be involved in the ubiquitination of a few MVB cargoes and lysosomal proteins [30,31].

The observation of Rsp5p-dependent ubiquitination at MVBs raises questions similar to those raised following observation of the role of this E3 in endocytic internalization. How are substrates recognized? With the exception of Sna3p, most of the known substrates do not carry PY motifs, and a requirement for PY-containing adaptors for Rsp5p recruitment has been predicted. Indeed, the PY-containing membrane protein Bsd2p, identified in a genetic screen for its role in trafficking of the manganese transporter Smf1p [32], is involved in the sorting of a few MVB cargoes, including Smf1p [33]. The correct sorting of Smf1p to MVBs involves the association of Bsd2p with two additional PY-containing membrane proteins, Tre1/2 [34].

Potential Rsp5p adaptors have recently been identified, based on the wealth of information deduced from genomic and proteomic approaches, and the use of protein microarray ubiquitination assays in vitro [35,36]. Ear1p (endosomal adaptor of Rsp5p), another endosomal PY-containing membrane protein, has been identified as both a binding partner and a substrate of Rsp5p. Deletion of EAR1, when combined with that of its redundant homologue, SSH4, led to a trafficking defect. In the double mutant the Rsp5p-dependent ubiquitination and MVB sorting of several cargoes are impaired, including Phm5p or transporters such as Gap1p and the siderophore transporter Sit1p, when directly routed to the endosomal pathway. No defect in Smf1p MVB sorting, which depends on the Bsd2/Tre1/2 adaptors [33,34], was observed. Other MVB cargoes, either directly fused to ubiquitin, or containing PY motifs enabling their direct interaction with and ubiquitination by Rsp5p, were also sorted to MVBs independently of Ear1p/Ssh4p. Thus Ear1p/Ssh4p appears to be essential for the MVB sorting of a specific set of cargoes by acting on their ubiquitination status [37]. It remains unclear how Ear1p and Ssh4p promote close connections between Rsp5p and its MVB substrates. Both proteins carry a B30.2/SPRY domain, known to be involved in protein–protein interactions [38] and that could be involved in this interaction. An alternative mechanism has been proposed for Bsd2p/Tre1/Tre2-dependent Smf1p ubiquitination. The Tre1/2 proteins recognize Smf1p, probably through interaction with polar residues in transmembrane regions, while simultaneously binding Bsd2p. Rsp5p could be brought into close contact with Smf1p through interaction of its WW domain with the PY motifs of Bsd2/Tre1/Tre2 [33]. Adaptors of Nedd4/Nedd4-like proteins probably play a similar role in mammals, as a number of PY-containing proteins, partners of Nedd4/Nedd4-like proteins, have been reported to be involved in the recruitment of these E3s to the Golgi apparatus/endosomes [39]. Interaction of the C2 domain of Rsp5p/Nedd4-like proteins with phospholipids, including phosphoinositides in particular, probably reinforces adaptor-mediated recruitment to a specific membrane [10,25].

What type of ubiquitination is associated with and required for MVB sorting? Cps1p was initially reported to undergo mono-ubiquitination. Ubiquitin (with its main target lysine mutated) fused in-frame to lysine-less Cps1p or Phm5p has been shown to restore MVB sorting [24,40]. These findings led to the hypothesis that mono-ubiquitination is the signal required for MVB sorting. However, ubiquitin-binding domains present in proteins of the ESCRT machinery have low affinity for mono-ubiquitin [41]. Plasma membrane proteins ubiquitinated at the cell surface by Rsp5p are still oligo-ubiquitinated when they arrive at the MVBs [42]. The MVB cargo Sna3p was recently reported to undergo Rsp5p-mediated polyubiquitination at a single target lysine, with up to seven or eight ubiquitin moieties linked via Lys-63 (Figure 1). This pattern of ubiquitination was altered in cells unable to form Lys-63-linked ubiquitin chains [11]. For another cargo, Sit1p, MVB sorting was profoundly affected in cells unable to form Lys-63-linked ubiquitin chains [19a]. However, this may be due to an indirect effect of these ubiquitin chains on the MVB machinery.

The observation that Rsp5p modifies some MVB and endocytic substrates with Lys-63-linked ubiquitin chains is consistent with the recent discovery that Rsp5p and the DUB Ubp2p are present within the same complex, and the demonstration that these enzymes preferentially assemble and disassemble respectively Lys-63-linked ubiquitin chains [20,43]. A requirement for Lys-63-linked ubiquitin chains at the MVB is also consistent with the demonstration that the mammalian DUB enzyme AMSH [associated molecule with the SH3 (Src homology 3) domain of STAM (signal-transducing adaptor molecule)], which specifically disassembles UbK63 chains [44], is partly endosomal, associates with ESCRT proteins and is activated by an ESCRT-0 component (STAM) [45].

Little is known about the mechanisms underlying the possible function of Lys-63-linked ubiquitin chains as internalization signals at the plasma membrane and MVBs, but several proteins carrying ubiquitin-binding domains, such as the UIM (ubiquitin-interacting motif) or UBA (ubiquitin-associated domain), play a key role in trafficking. Lys-63-linked ubiquitin chains have an open, linear conformation [46] in which the hydrophobic patch centred on ubiquitin Ile-44 is readily accessible for ligand binding. A clue to the possible role of these chains in trafficking is provided by the observation that the UBA domain of Ede1p, a protein involved in the internalization step of endocytosis, and the UIM domain of Hrs/Vps27 (vacuolar protein sorting 27), an ESCRT-0 component, have a specific or strong affinity for Lys-63-linked ubiquitin chains [41,47].

Rsp5p and Golgi-to-endosome sorting

In addition to its role in ubiquitin-mediated internalization at the cell surface and sorting to MVBs, a role for Rsp5p in the exit of transporters from the Golgi apparatus and their transfer to the endosomal pathway for vacuolar degradation has also been reported. Regulation of the internalization rate is not the only mechanism by which eukaryotic cells monitor the steady-state abundance of transporters at the plasma membrane. The fate of newly synthesized transporters within the secretory pathway can be regulated by diverting the protein to the lysosome/vacuole without passing by the plasma membrane, in response to environmental conditions. Typical physiological situations inducing this type of control mechanism include hormonal regulation, changes in substrate concentration or the availability of alternative nutrients. Examples in mammals include insulin-mediated trafficking of GLUT4 (glucose transporter 4). The first example identified in yeast was Gap1p, which is sorted to the vacuolar pathway in an Rsp5p-dependent manner when synthesized in the presence of a rich source of ammonium. Tat2p, another amino-acid transporter, displays a similar sorting pattern in the presence of high tryptophan concentrations. In an rsp5 mutant, Gap1p and Tat2p are recovered at the plasma membrane under conditions known to lead to their vacuolar sorting in wild-type cells. It was thus suggested that Rsp5p is required for the Golgi exit of these transporters, in addition to being required for internalization and sorting to MVBs (reviewed in [8]).

The role of ubiquitin and Rsp5p in the regulated sorting of plasma membrane transporters was recently reconsidered in the case of the siderophore transporter Sit1p. Newly synthesized Sit1p is sorted to the plasma membrane or to the endosomal/vacuolar pathway, depending on the presence or absence of its substrate, FOB (ferrioxamine B) [48]. After its production in the absence of FOB in rsp5Δ cells, Sit1p was recovered at the vacuolar membrane. Thus it was able to exit the Golgi apparatus, but was not sorted to MVBs. Kinetic experiments on hypomorphic rsp5 cells demonstrated that Sit1p exited the Golgi apparatus and was sorted to endosomes, but the impairment of MVB sorting resulted in a redistribution from endosomes to the plasma membrane [19a]. Consistent with the hypothesis that ubiquitination is not required for Golgi exit into the endosomal pathway, non-ubiquitinatable mutant forms of Cps1p, Phm5p and Sna3p are trapped at the vacuolar membrane. However, these proteins are not recovered at the plasma membrane. The plasma membrane redistribution of endosomal proteins appears therefore possible only for transporters [19,28], perhaps because they carry appropriate plasma membrane sorting signals.

Therefore ubiquitination does not appear essential itself for the exit from the Golgi apparatus of some MVB cargoes. However, Rsp5p has been shown to function at the Golgi/endosome interface when the intracellular fate of some MVB cargoes is analysed, including PY-containing proteins. This function of Rsp5p was clearly different from its enzymatic function at MVBs. Mutations impairing the interaction of Sna3p with Rsp5p led to deficiencies in the sorting of Sna3p, which was recovered in small structures (Golgi vesicles/endosomes?) upstream from MVBs, whereas a non-ubiquitinatable form of Sna3p was targeted to the vacuolar membrane [11]. The physical interaction between Sna3p and Rsp5p appears to be crucial for Sna3p sorting to the endosomal pathway. The function of Rsp5p in Sna3p sorting as a mere consequence of a strong interaction would be similar to that fulfilled by its homologue in humans, Nedd4, in the Golgi-to-lysosome trafficking of LAPTM5 (lysosome-associated protein transmembrane 5), which is sorted from the Golgi apparatus to lysosomes after the binding of its PY motifs to Nedd4 WW domains [31].

Conclusion and perspectives

Rsp5p plays a pleiotropic role in trafficking, and its functions are representative of the trafficking functions of Nedd4/Nedd4-like proteins in mammals [6,7]. The role of Rsp5p in sorting at the plasma membrane and MVBs is based principally on its function in ubiquitination. In addition, interaction with Rsp5p, apparently acting as a scaffold, seems to play a crucial role in the Golgi-to-endosome sorting of some PY-containing proteins. An increasing number of MVB cargoes of Rsp5p are thought to be recognized by PY-containing adaptors, and PY-containing proteins have also been shown to be required for the ubiquitination and internalization of a few plasma membrane proteins. The mechanisms of action of the PY-containing Rsp5p adaptors identified to date remain unclear. Depending on the cargo, an adaptor may function alone with a single substrate or in combination with other adaptors for another substrate. Additional adaptors will undoubtedly be discovered in the future, possibly from the long list of PY-containing proteins shown to interact with Rsp5p in genomic and proteomic studies [35,36]. The four Rsp5p partners shown to function as adaptors for the ubiquitination of MVB cargoes are themselves ubiquitinated and sorted to MVBs [34,37]. It remains unclear whether this modification is important for the function of these adaptors.

Consistent with studies showing a preference for Rsp5p in the assembly of Lys-63-linked ubiquitin chains [20,43], the list of endocytic and MVB cargoes modified by Lys-63-linked ubiquitin chains is continuing to increase [8,11,19,19a]. Similarly, the first example of a Nedd4 endocytic substrate displaying this type of ubiquitination has also been reported [6]. The structural features of Rsp5p/Nedd4 accounting for this preference towards the synthesis of UbK63-linked chains remain to be determined, as does the precise role of these chains in sorting processes. Rsp5p has also been described to promote mono-ubiquitination, and even the production of Lys-48-linked ubiquitin chains, ultimately leading to the proteasomal degradation of the target. How is the same E3 able to promote different types of ubiquitination? The mono-ubiquitination of some Rsp5p substrates, particularly endocytic substrates, may result from the trimming of pre-existing UbK63-linked ubiquitin chains by Ubp2p, which associates with Rsp5p and specifically disassembles this type of chain [43]. For Rsp5p- and Nedd4- or Nedd4-like-dependent polyubiquitination by Lys-63-, Lys-29- [49] or Lys-48-linked chains, it remains unclear whether the chain specification depends on the substrates themselves, requires the involvement of particular adaptors and/or E4, or the binding of preformed ubiquitin chains.

Finally, new functions of Rsp5p/Nedd4 E3s in trafficking are likely to emerge. Genetic and cell biology evidence has already been obtained for a link between Rsp5p and the actin cytoskeleton [50], and these findings are supported by proteomic evidence indicating that Rsp5p interacts with numerous proteins involved in actin cytoskeleton organization. Future investigations should improve our knowledge of the corresponding underlying functions.

Acknowledgments

We apologize to numerous colleagues who have been incompletely or not cited in this review because of space limitation. Our work on Rsp5p was supported by the CNRS, and the Université Paris Diderot/Paris 7, and by grants from the ARC (Association pour la Recherche contre le Cancer; grant 3298) and the EU (European Union) 6th Framework Programme [RUBICON (Role of Ubiquitin and Ubiquitin-like Modifiers in Cellular Regulation) NoE (Network of Excellence) and the Marie Curie research training network UBIREGULATORS]. S.L. is a Postdoctoral Fellow of the RUBICON programme, and Z.E. is a Postdoctoral Fellow of the UBIREGULATORS network.

Footnotes

  • Third Intracellular Proteolysis Meeting: A joint Biochemical Society and INPROTEOLYS Network Focused Meeting held at Auditorio de Tenerife, Santa Cruz de Tenerife, Canary Islands, Spain, 5–7 March 2008. Organized and Edited by Rosa Farràs (Centro de Investigación Príncipe Felipe, Valencia, Spain), Gemma Marfany (Barcelona, Spain), Manuel Rodríguez (CICbioGUNE, Derio, Spain), Eduardo Salido (La Laguna, Tenerife, Spain) and Dimitris Xirodimas (Dundee, U.K.).

Abbreviations: DUB, enzyme, deubiquitination enzyme; ESCRT, endosomal sorting complexes required for transport; FOB, ferrioxamine B; HECT, homologous to E6-associated protein C-terminus; MVB, multivesicular body; Nedd, neural-precursor-cell-expressed, developmentally down-regulated; npi, nitrogen permease inactivator; RING, really interesting new gene; STAM, signal-transducing adaptor molecule; UBA, ubiquitin-associated domain; UbK63, ubiquitin Lys-63; UIM, ubiquitin-interacting motif; Vps, vacuolar protein sorting

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