Ubiquitin in trafficking: The network at work

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Review

Ubiquitin in trafficking: The network at work Filippo Acconcia a , Sara Sigismund a , Simona Polo a,b,⁎ a

IFOM Foundation, The FIRC Institute for Molecular Oncology, Via Adamello 16, 20139 Milan, Italy Dipartimento di Medicina, Chirurgia ed Odontoiatria, Universita' degli Studi di Milano, Via di Rudini 8, 20122 Milan, Italy

b

A R T I C L E I N F O R M AT I O N

AB ST R AC T

Article Chronology:

Targeting of membrane proteins to their proper destination requires specific mechanisms. Protein

Received 6 October 2008

cargos are included in vesicles that bud off a donor organelle and ultimately fuse with a target

Accepted 15 October 2008

organelle, where the cargos are delivered. Endocytosis of transmembrane receptors (e.g., receptor

Available online 28 October 2008

tyrosine kinases, RTKs) follows a common scheme that consists of an internalization reaction and a delivery step, during which cargos are transferred to an endosomal station to be either directed to

Keywords:

the lysosome for degradation or recycled back to the cell surface. At each stage along the endocytic

Ubiquitin

route, short motifs within protein cargos and/or post-translational modifications regulate

Endocytosis

transmembrane receptor sorting. In recent years, studies have shown that ubiquitination acts as

Trafficking

a signal for the internalization and sorting of plasma membrane proteins. Here, we present an

Downmodulation

overview of ubiquitin's role as a ‘signal’ for intracellular trafficking and give examples of the

Signaling

multifaced mechanisms of ubiquitin-regulated RTK endocytosis.

Ubiquitin receptor

© 2008 Elsevier Inc. All rights reserved.

RTKs DUBs

Contents Role of Ub in transmembrane receptor internalization . . . . . . . . . . Regulation of endocytic proteins by monoubiquitination . . . . . . . . Role of Ub in sorting to multivesicular bodies and lysosome biogenesis Role of deubiquitination in receptor trafficking . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Modification by ubiquitin (Ub) remodels target proteins by affecting their stability, interaction with other proteins, enzymatic activity, and also their subcellular localization [1–3]. Ubiquitination occurs through the sequential activation of a cascade of reactions catalyzed by three classes of enzymes (E1, E2 and E3), resulting ultimately in the covalent attachment of Ub to the ɛ-amino group of

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a lysine (Lys) residue in the target protein. Often, several Ub moieties are added to the target protein in the form of polyubiquitin chains that can take on distinct topologies [4]. Indeed, all seven Lys residues present in an Ub molecule can be used for chain formation in vivo [5]. Different Ub-linkage topologies are associated with diverse biological functions [6]. For example, ubiquitin chains of at

⁎ Corresponding author. IFOM Foundation, The FIRC Institute for Molecular Oncology, Via Adamello 16, 20139 Milan, Italy. Fax: +39 02 574303231. E-mail address: [email protected] (S. Polo). 0014-4827/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2008.10.014

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least four subunits, linked via Lys48, can trigger degradation of the target protein by the 26S proteasome [4]. The functions of Lys63linked chains range from DNA damage tolerance and protein trafficking to ribosomal protein synthesis [4]. Much less is known about the roles of the other Ub-chain linkages [4,6]. Proteins can also be modified by monoubiquitination or multiubiquitination, i.e. the addition of a single, or multiple, monomeric Ub molecule(s), respectively. These regulatory signals have important roles in processes as diverse as endocytosis, virus budding, nuclear shuttling and transcription [1,2,7]. Removal and recycling of Ub are achieved by specific deubiquitinating enzymes (DUBs) that catalyze the removal of Ub-moieties from ubiquitinated substrates. DUBs serve to counterbalance ubiquitination reactions within a cell, thus dynamically contributing to the regulation of various cellular processes, such as endosomal sorting [8]. Ubiquitin alters the molecular landscape of the target protein, providing an additional surface for protein–protein interactions. Indeed, several Ub binding domains (UBDs) have been identified and characterized (reviewed in [9,10]). These protein modules, which do not display strict sequence conservation, interact with Ub via diverse three-dimensional folds and direct the flow of information to specific signaling pathways. The variety of structurally distinct Ub modifications, the dynamic and reversible nature of the ubiquitination cascade and the ability of UBDs to decipher, transduce and amplify the Ub-based signal, make Ub a highly versatile intracellular messenger [6]. The Ub network is of paramount importance in the physiological modulation of many cellular processes, including endocytosis of transmembrane receptors, which will be the focus of this review.

polyubiquitination of GPCR-associated β-arrestins has been proposed to be necessary for the internalization of receptors in clathrin-coated pits [15]. The role of ubiquitination in receptor tyrosine kinases (RTKs) internalization has been extensively studied. Indeed, platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) were the first mammalian receptors found to be ubiquitinated [17,18]. Since these studies, several other RTKs have been shown to be ubiquitinated in a ligand-dependent manner by the E3 ligase Cbl (see Table 1 and references therein). In the case of Met, the molecular mechanism of receptor ubiquitination has been investigated in detail. Stimulation of Met with hepatocyte growth factor (HGF) leads to detectable receptor ubiquitination and enhanced degradation [19,20]. The juxtamembrane region of Met contains a docking site for Cbl (phosphotyrosine 10 03), which is required for ligand-dependent ubiquitination and degradation of the receptor [20]; in keeping with this finding, this Cbl docking site is absent in the Tpr-Met oncogene [21]. Cbl can also be recruited to Met indirectly through the Grb2 adapter protein [20]. Park and co-workers have proposed a mechanism whereby the direct and indirect binding sites cooperate to position Cbl correctly on the receptor. Interestingly, this modality of Cbl recruitment could also be envisioned for other RTKs (e.g. EGFR, described below, and Ret [22]). Mutants of the

Table 1 – Tyrosine kinase receptors regulated by ubiquitination RTK

Role of Ub in transmembrane receptor internalization One of the first non-proteasomal functions discovered for Ub came from studies performed in yeast, in which Ub was shown to be a critical determinant for intracellular protein trafficking. Pioneering work in this model system demonstrated that Ub is required for the first step in cargo internalization, as well as for targeting cargos to vacuoles [11,12]. Indeed, monoubiquitination alone of several transmembrane receptors (alpha-factor receptors, permeases and transporters) is sufficient to trigger their internalization, although modification with Lys63-linked Ub chains will speed up this process [13]. In mammalian cells, the situation is more complex since not only the receptor, but also the endocytic adaptors, are often ubiquitinated in response to extracellular stimuli. Furthermore, more than one internalization pathway exists in the cell and not all of them appear to be regulated by Ub. Early studies on the growth hormone receptor (GHR) have shown that although ubiquitination of the receptor itself is not essential for internalization, an intact ubiquitination machinery is required [14]. The same is also true for a class G protein-coupled Receptors (GPCRs) that are ubiquitinated upon agonist stimulation [15]. In the case of the β2-adrenergic receptor and the chemokine receptor, CXCR4, mutation of all the lysine residues in the cytoplasmic tail did not affect the initial internalization event, but severely impaired the downstream endocytic sorting step that targets receptors to the degradative pathway [15,16]. In this case,

E3-Ligase

EGFR

Cbl

VEGFR-1 VEGFR-2

Cbl Cbl Nedd4 Cbl

PDGFR FGFR IGF-1R

Met/ HGFR TrkA ErbB3 ErbB4 c-Kit TGFβ

Cbl Nedd4 Mdm2 Cbl Cbl TRAF6 Nedd4-2 NRDP1 NRDP1 AIP1/Itch Cbl

Ret

Nedd4-2 Smad7/ Smurf2 WWP1/ Tiul1 Cbl

CSF-1R

Cbl

Type of Ub-modification

References

MultimonoUb PolyUb Lys63 ? ?

Haglund et al. [25] Huang et al. [26] Kobayashi et al. [87] Duval et al. [83] Murdaca J., JBC 2004 Joazeiro et al. [86] Haglund et al. [25] Wong et al. [97] Vecchione et al. [96] Girnita et al. [85] Sehat et al. [33] Peschard et al. [20] Carter et al. [82] Geeta et al. [84] Arevalo et al. [38] Qiu and Goldberg [93] Omerovic et al. [92] Sundvall et al. [94] Zeng et al. [98] Masson et al. [91] Suzuki et al. [95] Komuro et al. [88] Kuratomi et al. [89]

MultimonoUb PolyUb ? ? PolyUb Lys63 PolyUb Lys48 MultimonoUb PolyUb Lys48 PolyUb Lys63 MultimonoUb PolyUb PolyUb MonoUb ? PolyUb

? MonoUb/PolyUb ?

Question mark means “not characterized”.

Scott et al. [22] Lee et al. [90]

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Met receptor that are insensitive to Cbl-dependent ubiquitination are transforming and tumorigenic due to their enhanced stability and the sustained activation of downstream signaling pathways [19,20]. As in the case of Met, ubiquitination has been shown to play an essential role in the downmodulation of the majority of RTKs (for a recent review see [23]). However, the situation is less clear for the requirement of Ub in the initial step of endocytosis. Indeed, recent studies have brought into question the involvement of Ub in receptor internalization. This is the case of Fibroblast Growth Factor Receptor (FGFR) [24] and EGFR (see below), where mutation of the major ubiquitination sites does not affect internalization. Ligand-induced EGFR trafficking (i.e., internalization and sorting to the lysosome) is one of the best-characterized examples of how the regulation of receptor turnover is exquisitely modulated by the Ub signal. Initial studies based on the use of a chimeric protein composed of EGFR and an Ub mutant that could not be extended by polyubiquitination, showed that a single Ub was sufficient to drive internalization, although at a lower rate compared to wild-type receptor [25]. More recently, analysis by quantitative mass-spectrometry has revealed that EGFRs are both mono- and polyubiquitinated through Lys63-linked chains [26]. These data suggest that although monoUb is sufficient for internalization, as observed in yeast, polyubiquitination through Lys63 generates a more efficient internalization signal, possibly by increasing the avidity of binding to UBD-containing proteins. Another possibility, that needs to be verified experimentally, is that monoUb and Lys63-linked chains represent distinct signals acting at different steps along the endocytic route. Recent studies have shown that EGFR ubiquitination is not essential for internalization through clathrin-coated pits. Indeed, EGFR mutants defective in ubiquitination, either mutagenized in the E3-ligase binding site or in the acceptor lysines for Ub, present no major internalization defects [27–29]. This apparent discrepancy between ubiquitination being sufficient, but not required, for EGFR internalization can be explained by the existence of alternative internalization pathways possibly regulated by different signals (beside Ub, the AP-2 recognition motif and multiple phosphorylated tyrosines present in receptor tail [30]). We have shown that the level of EGFR ubiquitination, regulated by ligand concentration, correlates with the differential recruitment of the EGFR into distinct endocytic pathways [28,31] (Fig. 1A). At low EGF doses, receptor ubiquitination is not detected and the EGFR is internalized through clathrinmediated endocytosis only, while at high EGF doses, both clathrindependent and -independent internalization routes come into play as the receptor becomes ubiquitinated. Indeed, ubiquitination appears to be required for clathrin-independent endocytosis while it is dispensable for clathrin-dependent internalization [28]. Preliminary observations suggest the existence of a cooperative mechanism controlling receptor ubiquitination, which displays sigmoidal behaviour as a function of EGF dose (SS and SP unpublished results). One possible explanation for the threshold in EGFR ubiquitination could reside in the mechanism of Cbl recruitment to the receptor. Indeed, similarly to Met, multiple direct and indirect (through Grb2) binding sites for Cbl are present in the receptor tail of EGFR and these sites might cooperate in E3 binding. The existence of distinct endocytic pathways raises a number of questions. Are all endocytic routes equivalent from a functional point of view? Or are they associated with different receptor

functions? Finally, if endocytosis is required for both receptor attenuation and signaling, how are these two opposing outcomes coordinated? Interestingly, the majority of EGFRs internalized via clathrin-mediated endocytosis are not targeted to degradation, but rather are recycled to the cell surface [32] (Fig. 1A). Conversely, clathrin-independent internalization preferentially commits the receptor to degradation [32]. This has profound implications for signaling, as by skewing EGFR fate towards recycling rather than degradation, clathrin-mediated endocytosis prolongs the duration of the signal [32]. Thus, Ub might play a critical role in deciding between receptor signaling and downmodulation, already at the internalization step. However, this hypothesis requires further experimental validation. These findings indicate that cells are able to sense growth factor concentration and to decode the information stored in the stimulus' strength, converting it to different outputs (e.g. distinct ubiquitination patterns, distinct endocytic routes and, finally, distinct biological responses). A similar mechanism has been recently described for IGF-1R [33] (see also Leon and HaguenuarTsapis in this issue) and PDGFR [34]. In the latter case, cells switch from a migrating to a proliferating phenotype in response to an increasing PDGF gradient. It was proposed that the decision to proliferate or migrate relies on the distinct endocytic route followed by the receptor in response to ligand concentration [34]. The idea that cells can execute distinct biological responses depending on ligand concentration is not novel; e.g. during development, gradients of morphogens are established that can induce distinct signaling events in cells positioned differently along the gradient [35]. Emerging evidence points to an essential role of receptor trafficking also in the regulation of this kind of response [35]; however, how cells sense ligand concentration and translate it into a qualitative output is still not understood and certainly deserves further investigation. A similar correlation between the ubiquitination status of the receptor and its recruitment to distinct endocytic routes, which lead to different receptor fates, has previously been described for the TGFβ receptor [36]. When the TGFβ receptor is associated with the Smad7–Smurf2 E3 ligase complex, it is recruited to caveolae and, after internalization, is directed to degradation. In contrast, when the receptor is associated with Smad2 and SARA (Smad Anchor for Receptor Activation), it is internalized via clathrinmediated endocytosis and signaling proceeds from the endosomal compartment [36] (Fig. 1B). Recent studies in Xenopus suggest that an additional level of regulation in TGFβ receptor endocytosis is achieved downstream of the plasma membrane by the small GTPase Rap2 (Fig. 1B), which competes with Smad7 for binding to the receptor, diverting the fate of the receptor from a degradative to a recycling/signaling fate [37]. A clear role for Ub in signaling was demonstrated in the case of TrkA, a receptor for the nerve growth factor (NGF) [38,39]. Indeed, TrkA internalization in distal axonal processes is required for retrograde signaling to neuronal soma, which ultimately controls neuronal differentiation [40]. Two reports have shown that TrkA receptor is ubiquitinated upon NGF stimulation [38,39]. In one report, the authors proposed that ubiquitination is mediated by interactions with the p75 neurotrophin receptor and its associated E3 ligase, TRAF6, which catalyzes Lys63-linked polyubiquitination of TrkA [39]. In the second report, Nedd4-2 was proposed as the E3 ligase for NGF-dependent multi-monoubiquitination of TrkA [38]. More work is needed to understand how these findings can be

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Fig. 1 – Multiple sorting steps control EGFR and TGFβR trafficking and signaling. (A) The first sorting step is at the plasma membrane, where EGFR can be internalized through different endocytic pathways as a function of EGF dose. In the clathrin route, receptors are mostly directed to recycling and signaling, while in the non-clathrin route they are preferentially targeted for lysosomal degradation. A second sorting step is present at the level of the endosomes, where the two internalization pathways seem to converge. A flat clathrin lattice on the endosomal membrane stabilizes the interaction between the ESCRT-0 complex (Hrs, STAM and Eps15b) and ubiquitinated EGFR, which is then targeted for degradation. Counteraction between DUBs (possibly AMSH) and Cbl is shown. (B) TGFβRs internalized through clathrin-mediated endocytosis are directed towards the early endosomes (enriched in PIP3, phosphatidylinositol-3-phosphate). Here, interaction with the SARA/Smad2 complex allows signaling and recycling. In the caveolar pathway, TGFβRs associate with the SMAD7–SMURF2 complex, which targets receptors for ubiquitin-dependent degradation. In this route, TGFβRs reach a yet undefined compartment, which is in dynamic communication with the “SARA signaling endosome”. A second sorting step at this level is exerted by the small-GTPase Rap2, which counteracts the action of Smad7, leading to delayed receptor degradation and increased signaling/recycling.

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integrated into a comprehensive picture. In both studies, receptor ubiquitination was shown to be critical for TrkA endocytosis and signaling, but whether Ub is required for the internalization step remains unclear. Another example of how Ub is a critical regulator of signaling is represented by the cell fate determination Notch pathway (extensively reviewed elsewhere, see [41,42]). In conclusion, ubiquitination (i.e., monoubiquitination or Lys63-linked polyubiquitination) regulates the internalization of a given cargo in different ways, depending on the receptor system. The different topology of the Ub-based signals may be important for the fine modulation of the receptor endocytic entry route, which is ultimately critical for the balance between receptorinduced signal transduction and receptor downmodulation.

Regulation of endocytic proteins by monoubiquitination Ubiquitin-dependent internalization of receptors requires the precise molecular recognition of the ubiquitinated cargo by UBD-containing proteins (i.e., Ub receptors). Since UBDs bind to mono- and poly-Ub with low affinity, usually in the micromolar range (for reviews see [9,10]), a single Ub:UBD interaction would not be sufficient for the efficient internalization of the ubiquitinated receptor. Nonetheless, several mechanisms exist to transform low-affinity contacts into physiologically relevant interactions. Ub receptors, such as the endocytic adaptors eps15 or epsin, contain multiple UBDs as well as additional protein modules that recognize other endocytic proteins or membraneassociated phosphoinositides. The presence of multiple UBDs allows the Ub receptor to either bind several monoUb molecules in multi-monoubiquitinated cargos [9] or to engage a single monoUb through different UBDs [43,44], thus increasing binding affinity. Alternatively, endocytic adaptors (Ub receptors) may be preferentially targeted to their ubiquitinated cargos by binding simultaneously to Ub (in the cargo) and to the plasma membrane [2]. Interestingly, an increase in the binding avidity between Ub receptors and ubiquitinated proteins can also be achieved by a linear topology of Lys63-linked polyubiquitination. Many Ub receptors themselves undergo monoubiquitination through a molecular process that requires an intact UBD (i.e., coupled monoubiquitination) [10]. Recently, the molecular mechanism of coupled monoubiquitination has been clarified using eps15 as a model system [31]. It involves the interaction between a “competent” Ub interacting motif (UIM) in the Ub receptor and a HECT-type E3 ligase (Nedd4, in the case of eps15), which has been itself modified by ubiquitination. This modified E3 enzyme is then able to transfer another thiolester-conjugated Ub from the catalytic Cys residue to the substrate [31]. In an analogous manner, the Ub-like (Ubl) domain of a RING-type Ub ligase (e.g., Parkin) interacts with the UIM of the Ub receptor and mediates its monoubiquitination by catalyzing the transfer of Ub directly from the E2 conjugating enzyme to the substrate [45]. A variation on theme is when an Ub receptor can bind directly to the E2 conjugating enzyme via an interaction between its UBD and Ub linked to E2 via a thioester bond [46]. Both modes of ubiquitination, E3-independent and E3-dependent, can, in principle, coexist: E3independent ubiquitination may control the level of constitutive monoubiquitination of UBD-containing proteins, whereas, E3dependent ubiquitination may be responsible for monoubiquitina-

tion in physiologically relevant signaling-regulated processes. Whether constitutive E3-independent monoubiquitination of such Ub receptors is important for regulating endocytosis remains to be determined. A major open issue in the field concerns the functional role of the monoubiquitination of endocytic proteins. Among others, two hypotheses are most plausible. On one hand, coupled monoubiquitination would extend the range of intermolecular interactions of the Ub receptor, creating a network of Ub-mediated interactions, leading to signal amplification and progression of ubiquitinated cargos along the endocytic pathway [6]. On the other hand, intramolecular interactions between monoUb and UBDs within the endocytic adaptor may lead to an autoinhibitory mechanism, which causes the dissociation of the Ub receptor from the ubiquitinated cargo (e.g., Sts2 and ubiquitinated EGFR) [2,47]. These two possibilities are not mutually exclusive and both mechanisms could be involved in the regulation of endocytic processes, possibly by acting at distinct trafficking steps and/or regulating different endocytic adaptors.

Role of Ub in sorting to multivesicular bodies and lysosome biogenesis Biosynthetic and endocytic pathways converge at the level of the early endosomes, which contain proteins coming from the trans Golgi network (TGN) and the plasma membrane. In early endosomes, nonubiquitinated proteins are recycled to the plasma membrane or directed to other intracellular compartments. In contrast, ubiquitinated proteins are sorted into intraluminal vesicles (ILVs), thereby generating the so-called multivesicular bodies (MVB) [48,49]. Aside from plasma membrane receptors destined for degradation, lysosomal integral membrane glycoproteins (referred to as LAMPs), enzymes such as precursor carboxypeptidase S, and ubiquitinated transporters are diverted from their normal route after exit from the Golgi compartment to late endocytic compartments (see also Thomas Falguieres, PierrePhilippe Luyet and Jean Gruenberg in this issue). Ground-breaking work from Scott Emr's lab, based on genetic selections in Saccharomyces cerevisiae, has defined a large number of vacuolar protein sorting (vps) mutants, defective in the delivery of proteins to lysosomes/vacuoles [50]. Thanks to this work and to the subsequent functional analysis of vps mutants performed by several groups, the molecular machinery involved in this second sorting step has largely been identified and ubiquitination was found to be critical for the activation of the degradative MVB pathway [2]. Sorting of endosomal proteins into ILVs of MVBs is mediated by the endosomal sorting complexes required for transport (ESCRTs), which lie at the heart of a vast protein–protein and protein–lipid interaction network. Four ESCRTs have been so far identified (for an extensive review on this topic see [51], see also Harald Stenmark in this issue). ESCRT-0, -I and -II are engaged early in the sorting process and all three complexes contain protein modules capable of binding Ub. ESCRT-III, the final complex in the pathway, plays a role in the incorporation of cargo in ILVs. The initial step in sorting proteins to MVBs involves the recognition of Ub on receptors or cargos by ESCRT-0, which is composed by 3 proteins, Hrs (Vps27 in yeast), STAM, and eps15b, all possessing functional UBDs [51]. Hrs then binds to another Ub

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receptor, TSG101, which together with Vps28 and Vps37 forms the ESCRT-I complex. Progression through the sorting pathway from ESCRT-I to ESCRT-II also requires direct contact between ESCRTs components, as well as the binding of ubiquitinated cargo to specific UBD-containing proteins. In the case of ESCRT-II, the GLUEdomain of Vps36, which binds both Ub and membrane lipids, is critical for capturing ubiquitinated cargo [52]. ESCRT-III does not contain any UBD-containing proteins, but instead recruits a DUB, which removes Ub from the cargo before its inclusion into the lumen of the MVB [51] (see also the next section). It still remains to be determined how ESCRTs actually promote the invagination of the endosomal membrane and the scission of ILVs. Importantly, components of ESCRT-III have been shown to be required for the budding of ILVs (reviewed in [53]). These components normally exist in an auto-inhibited state in the cytosol; removal of autoinhibition induces membrane targeting and ESCRT-III assembly into a putative protein lattice [53]. Although the mechanism of polymerization of ESCRT-III proteins is still a matter of investigation [54–57], emerging evidence suggests a broader role for these proteins in regulating different aspects of cell physiology [58]. Indeed, ESCRT-III is required for processes topologically similar to ILV generation, such as the budding of enveloped viruses [59,60] and, as recently suggested, cytokinesis [58,59]. Stable or transient interference of components (e.g., Hrs, TSG101, Vps36 and Vps24) of ESCRTs has revealed that not all components are essential for sorting a given membrane receptor [51]. For example, while degradation of activated EGFR is completely prevented by Vps24 depletion, a component of the ESCRT-III complex, depletion of Hrs [61] or TSG101 [62,63] only partially impairs EGFR degradation. In addition, conflicting results over the requirement of ESCRT-II for lysosomal degradation of the EGFR have been reported [64,65]. However, subtraction of the same ESCRT-II does not affect the degradation of the MHC-I complex, but strongly inhibits ligand-induced downregulation of the cell surface receptor, ferroportin [64,65]. Therefore, it is possible that specific MVB components might come into play once different cargos reach the lysosomal compartment.

Role of deubiquitination in receptor trafficking Deubiquitination of cargos, as well as of proteins of the endocytic machinery, critically impacts on the regulation of intracellular trafficking. The first role discovered for DUBs in trafficking was the removal of Ub from cargos prior to their translocation into the lumen of MVBs [66]. This is an essential step in sorting, which ensures recycling of Ub in order to preserve intracellular Ub concentration. In yeast, this deubiquitination step is carried out by the DUB hydrolase, Doa4 [66], while in mammals the situation is more complex: two DUBs, USP8 (Ub Specific Protease 8, also called UBPY) and AMSH (Associated Molecule with the SH3 domain of STAM), which are recruited to endosomes by interactions between their N-terminal MIT domains and late-acting ESCRT-III subunits [53], can deubiquitinate cargos at this stage and direct them to quite different end-fates (see [8] for details). Recent data have established crucial, but distinct, roles for these two DUBs in the fine regulation of EGFR degradation. AMSH rescues EGFR from sorting to MVBs and consequently degradation, by removing Ub from the receptor at early stages of endosomal

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sorting, thus promoting its recycling [64,67,68]. AMSH might also act at a later step of MVB sorting, by competing with Vps4 for ESCRT-III binding, possibly inhibiting vesicles budding [8]. In contrast, UBPY activity seems to be required for the final commitment of the EGFR to MVBs and degradation, similarly to Doa4 in yeast. To exert this function, UBPY acts on multiple substrates along the sorting pathway, targeting the receptor itself, as well as endocytic adaptors and components of the ESCRT machinery, e.g., STAM [64,69,70]. Acting at different steps along the EGFR route to lysosomes, these DUBs balance the fate of EGFR between downregulation and recycling [71]. Interestingly, AMSH can process Lys63-linked, but not Lys48linked, polyubiquitin chains in vitro, and a recent crystallographic study has revealed the structural basis for this cleavage specificity [67–69,72]. These results reinforce the idea that AMSH is indeed involved in lysosomal but not proteasomal-dependent pathways. On the contrary, UBPY shows dual specificity for both Lys63- and Lys48-linked chains, suggesting a broader role for this DUB in both lysosomal and proteasomal degradation [69,73]. Other DUBs may regulate endocytic traffic of specific cargos at the level of the plasma membrane. In yeast, overexpression of an active Ubp1 disrupts lysosomal trafficking of the Ste6 transporter protein, as well as the α-factor receptor Ste2, without affecting their ubiquitination status [74]. Another DUB, UCH37, has been implicated in the regulation of the type-I TGFβ receptor degradation. In this case, however, the intracellular compartment where UCH37 acts and its possible cleavage specificity still remain to be understood [75]. A relatively unexplored field regards the identification of DUBs that act on monoubiquitinated endocytic proteins. Initial observations made in Drosophila melanogaster pointed to a regulatory role for the DUB fat facet (faf) on the stability of liquid facet (lqf), a homologue of the mammalian endocytic protein, epsin [76]. Faf, by preventing lqf proteasomal degradation, results in the correct development of the Drosophila eye [77,78]. Subsequent studies in mammalian cells confirmed the functional interaction between epsin and the mammalian homologue of faf, FAM/USP9X [79]. Although it was proposed that the FAM-dependent regulation of epsin monoubiquitination might impact on the ability of espin1 to interact with specific binding partners (e.g., AP-2, clathrin), the role of FAM/USP9x in receptor endocytosis is still unclear. Interestingly, FAM/USP9x colocalizes with different markers of the TGN and late endosomes [80]. Many questions about DUBs remain to be answered, mainly concerning their regulation and how substrate specificity is achieved. Further studies on the subcellular localization of DUBs and their substrates/binding partners will be key to understanding the function of these crucial enzymes in receptor trafficking and other cellular pathways.

Concluding remarks The role of ubiquitination in directing proteins from the limiting membrane of endosomes to MVB vesicles is well established. The efficiency of this sorting system is reflected by the fact that several viruses hijack this pathway in order to escape from cells once replicated. ILV formation is peculiar in that membranes must curve and bud away from (rather than into) the cytoplasm. Virus budding has the

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same topological requirements and this seems to explain why viruses have appointed the cellular MVB machinery to bud from cells. Accordingly, several studies have now established that the entire protein network required for human MVB biogenesis participates in the release of HIV and probably many other viruses (for a review see [81]). Viruses also encode their own ubiquitination enzymes that hijack the cellular Ub system to drive viral replication, by influencing cell proliferation, apoptosis and recognition of infected cells by the immune system. Retroviruses have historically served as important model systems for studying oncogenes and cancer and it is easy to envision that they could provide new insights into Ub-mediated trafficking processes. An in-depth analysis of this system should increase our molecular understanding of how endocytosis participates in the control of “effector” signaling and potentially provide new therapeutic targets for the treatment of pathological conditions in which Ub-mediated processes are altered.

Acknowledgments We apologize for having been unable to cite original reports due to space limitation. We thank Rosalind Gunby for critically reading the manuscript. Work in our laboratory is supported by grants from AIRC (Italian Association for Cancer Research) and AICR (Association for International Cancer Research). F.A. is supported by a fellowship from AIRC (Italian Association for Cancer Research).

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