Opposing activities of the Snx3-retromer complex and ESCRT proteins mediate regulated cargo sorting at a common endosome

Endocytosed proteins are either delivered to the lysosome to be degraded or are exported from the endosomal system and delivered to other organelles. Sorting of the Saccharomyces cerevisiae reductive iron transporter, composed of the Fet3 and Ftr1 proteins, in the endosomal system is regulated by available iron; in iron-starved cells, Fet3-Ftr1 is sorted by Snx3/Grd19 and retromer into a recycling pathway that delivers it back to the plasma membrane, but when starved cells are exposed to iron, Fet3-Ftr1 is targeted to the lysosome-like vacuole and is degraded. We report that iron-induced endocytosis of Fet3-Ftr1 is independent of Fet3-Ftr1 ubiquitylation, and after endocytosis, degradation of Fet3-Ftr1 is mediated by the multivesicular body (MVB) sorting pathway. In mutant cells lacking any component of the ESCRT protein-dependent MVB sorting machinery, the Rsp5 ubiquitin ligase, or in wild-type cells expressing Fet3-Ftr1 lacking cytosolic lysyl ubiquitin acceptor sites, Fet3-Ftr1 is constitutively sorted into the recycling pathway independent of iron status. In the presence and absence of iron, Fet3-Ftr1 transits an endosomal compartment where a subunit of the MVB sorting receptor (Vps27), Snx3/Grd19, and retromer proteins colocalize. We propose that this endosome is where Rsp5 ubiquitylates Fet3-Ftr1 and where the recycling and degradative pathways diverge.     

Internalization and down-regulation of the human epidermal growth factor receptor are regulated by the carboxyl-terminal tyrosines

The C terminus of the epidermal growth factor receptor (EGF-R) contains three tyrosines (Y1068, Y1148, and Y1173) which correspond to the major autophosphorylation sites. To investigate the role of the tyrosines in internalization and down-regulation of the EGF-R, mutational analysis was performed with receptors in which 1, 2, or all 3 tyrosines were changed to phenylalanines. The triple point mutant EGF-R, expressed in NIH-3T3, exhibited low autophosphorylation in vivo, low biological and reduced kinase activities. Single and double point mutants were down-regulated, as well as wild type EGF-R in response to EGF showing a half-life of about 1 h. Degradation of the triple point mutant, however, was impaired and resulted in a half-life of 4 h in the presence of EGF. EGF-dependent down-regulation of surface receptors was decreased in the triple point mutant EGF-R as was internalization and degradation of EGF. The specific rate of internalization of the triple point mutant was reduced. By contrast, intracellular processing of ligand previously internalized at 20 degrees C was similar between wild type and mutant receptors. Taken together the data indicate that the delay in degradation observed in cells expressing the triple point mutant EGF-R can be attributed mainly to a slower removal from the cell surface. Our results show that in the full-length EGF-R all three C-terminal tyrosines are necessary for rapid internalization, suggesting that autophosphorylation is required for efficient EGF-dependent receptor endocytosis.     

Annexin I is phosphorylated in the multivesicular body during the processing of the epidermal growth factor receptor

We have previously shown that an active epidermal growth factor receptor (EGF-R) kinase is necessary for efficient sorting of the EGF-R to the lysosome, and we have shown that this occurs in the multivesicular body (MVB), where EGF-R are sorted away from recycling receptors by being removed to the internal vesicles of the MVB. The aim of the present study was to identify substrates of the EGF-R kinase associated with MVBs which might play a role in this sorting process. We used a density shift technique to isolate MVBs and show that the major substrates phosphorylated in vitro within MVBs which contain an active EGF-R kinase are the EGF-R itself and annexin I. Annexin I is associated with both plasma membrane and MVBs in a calcium-independent manner but can be phosphorylated in vitro only in MVBs. Phosphorylation of calcium-independent annexin I in isolated MVBs converts it to a form that requires calcium for membrane association. In cells with an active EGF-R kinase the amount of calcium-independent annexin I in MVBs is reduced, suggesting that a phosphorylation-induced conversion of the calcium independent to the calcium-dependent form also occurs in vivo. Our observations, together with the known properties of annexin I in mediating membrane fusion, suggest that inward vesiculation in MVBs is induced by the EGF-R and is mediated by phosphorylated annexin I.     

Permease recycling and ubiquitination status reveal a particular role for Bro1 in the multivesicular body pathway

Ubiquitination of the yeast Gap1 permease at the plasma membrane triggers its endocytosis followed by targeting to the vacuolar lumen for degradation. We previously identified Bro1 as a protein essential to this down-regulation. In this study, we show that Bro1 is essential neither to ubiquitination nor to the early steps of Gap1 endocytosis. Bro1 rather intervenes at a late step of the multivesicular body (MVB) pathway, after the core components of the endosome-associated ESCRT-III protein complex and before or in conjunction with Doa4, the ubiquitin hydrolase mediating protein deubiquitination prior to their incorporation into MVB vesicles. Bro1 markedly differs from other class E vacuolar protein sorting factors involved in MVB sorting as lack of Bro1 leads to recycling of the internalized permease back to the plasma membrane by passing through the Golgi. This recycling seems to be accompanied by deubiquitination of the permease and unexpectedly requires a normal endosome-to-vacuole transport function.     

Separate endocytic pathways of kinase-defective and -active EGF receptor mutants expressed in same cells

Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.     

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of beta2-adrenergic receptors

Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated beta(2)-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate beta(2)-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the beta-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of beta(2)-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of beta(2)-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of beta(2)-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.     

The role of ubiquitination in lysosomal trafficking of δ-opioid receptors

The δ-opioid receptor (DOR) undergoes ligand-induced downregulation by endosomal sorting complex required for transport (ESCRT)-dependent endocytic trafficking to lysosomes. In contrast to a number of other signaling receptors, the DOR can downregulate effectively when its ubiquitination is prevented. We explored the membrane trafficking basis of this behavior. First, we show that internalized DORs traverse the canonical multivesicular body (MVB) pathway and localize to intralumenal vesicles (ILVs). Second, we show that DOR ubiquitination stimulates, but is not essential for, receptor transfer to ILVs and proteolysis of the receptor endodomain. Third, we show that receptor ubiquitination plays no detectable role in the early sorting of internalized DORs out of the recycling pathway. Finally, we show that DORs undergo extensive proteolytic fragmentation in the ectodomain, even when receptor ubiquitination is prevented or ILV formation itself is blocked. Together, these results are sufficient to explain why DORs downregulate effectively in the absence of ubiquitination, and they place a discrete molecular sorting operation in the MVB pathway effectively upstream of the ESCRT. More generally, these findings support the hypothesis that mammalian cells can control the cytoplasmic accessibility of internalized signaling receptors independently from their ultimate trafficking fate.     

Binding of filamin to the C-terminal tail of the calcitonin receptor controls recycling

Many G protein-coupled receptors undergo endocytosis, but the mechanisms involved in endocytic sorting and recycling remain to be fully elucidated. We found that the G protein-coupled calcitonin receptor (CTR) undergoes tonic internalization and accumulates within the cell. Using a fluorescence loss in photobleaching assay, we classified these vesicles functionally as recycling vesicles. In a two-hybrid screening, we found that the actin-binding protein filamin interacted with the C-terminal tail of the CTR. The degradation of the receptor was profoundly increased in the absence of filamin or the CTR-filamin interaction. The absence of filamin was also associated with a marked decrease in recycling of the receptor from the endosomes to the cell surface. In contrast, calcitonin-induced inhibition of spontaneous filamin proteolysis was associated with increased recycling of the receptor to the cell surface and decreased degradation of the CTR, suggesting an important role for filamin in the endocytic sorting and recycling of the internalized CTR.     

ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor-regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III-dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4-ESCRT-III interacting protein, bound to a YPX(3)L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX(n)L motifs.     

Identification of a novel endocytic recycling signal in the D1 dopamine receptor

A critical event determining the functional consequences of G protein-coupled receptor (GPCR) endocytosis is the molecular sorting of internalized receptors between divergent recycling and degradative membrane pathways. The D1 dopamine receptor recycles rapidly and efficiently to the plasma membrane after agonist-induced endocytosis and is remarkably resistant to proteolytic down-regulation. Whereas the mechanism mediating agonist-induced endocytosis of D1 receptors has been investigated in some detail, little is known about how receptors are sorted after endocytosis. We have identified a sequence present in the carboxyl-terminal cytoplasmic domain of the human D1 dopamine receptor that is specifically required for the efficient recycling of endocytosed receptors back to the plasma membrane. This sequence is distinct from previously identified membrane trafficking signals and is located in a proximal portion of the carboxyl-terminal cytoplasmic domain, in contrast to previously identified GPCR recycling signals present at the distal tip. Nevertheless, fusion of this sequence to the carboxyl terminus of a chimeric mutant delta opioid neuropeptide receptor is sufficient to re-route internalized receptors from lysosomal to recycling membrane pathways, defining this sequence as a bona fide endocytic recycling signal that can function in both proximal and distal locations. These results identify a novel sorting signal controlling the endocytic trafficking itinerary of a physiologically important dopamine receptor, provide the first example of such a sorting signal functioning in a proximal portion of the carboxyl-terminal cytoplasmic domain, and suggest the existence of a diverse array of sorting signals in the GPCR superfamily that mediate subtype-specific regulation of receptors via endocytic membrane trafficking.     

Rab15 effector protein: a novel protein for receptor recycling from the endocytic recycling compartment

Sorting endosomes and the endocytic recycling compartment are critical intracellular stores for the rapid recycling of internalized membrane receptors to the cell surface in multiple cell types. However, the molecular mechanisms distinguishing fast receptor recycling from sorting endosomes and slow receptor recycling from the endocytic recycling compartment remain poorly understood. We previously reported that Rab15 differentially regulates transferrin receptor trafficking through sorting endosomes and the endocytic recycling compartment, suggesting a role for distinct Rab15-effector interactions at these endocytic compartments. In this study, we identified the novel protein Rab15 effector protein (REP15) as a binding partner for Rab15-GTP. REP15 is compartment specific, colocalizing with Rab15 and Rab11 on the endocytic recycling compartment but not with Rab15, Rab4, or early endosome antigen 1 on sorting endosomes. REP15 interacts directly with Rab15-GTP but not with Rab5 or Rab11. Consistent with its localization, REP15 overexpression and small interfering RNA-mediated depletion inhibited transferrin receptor recycling from the endocytic recycling compartment, without affecting receptor entry into or recycling from sorting endosomes. Our data identify REP15 as a compartment-specific protein for receptor recycling from the endocytic recycling compartment, highlighting that the rapid and slow modes of transferrin receptor recycling are mechanistically distinct pathways.     

Fission of tubular endosomes triggers endosomal acidification and movement

The early endosome acts as a sorting station for internalized molecules destined for recycling or degradation. While recycled molecules are sorted and delivered to tubular endosomes, residual compartments containing molecules to be degraded undergo "maturation" before final degradation in the lysosome. This maturation involves acidification, microtubule-dependent motility, and perinuclear localization. It is currently unknown how sorting and the processes of maturation cooperate with each other. Here, we show that fission of a tubular endosome triggers the maturation of the residual endosome, leading to degradation. Use of the dynamin inhibitor dynasore to block tubular endosome fission inhibited acidification, endosomal motility along microtubules, perinuclear localization, and degradation. However, tubular endosome fission was not affected by inhibiting endosomal acidification or by depolymerizing the microtubules. These results demonstrate that the fission of recycling tubules is the first important step in endosomal maturation and degradation in the lysosome. We believe this to be the first evidence of a cascade from sorting to degradation.     

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.     

The deubiquitinases USP33 and USP20 coordinate β2 adrenergic receptor recycling and resensitization

Agonist‐induced ubiquitination of the β₂ adrenergic receptor (β₂AR) functions as an important post‐translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin‐specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late‐endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the β₂AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a ‘trip switch’ between degradative and recycling pathways at the late‐endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post‐endocytic sorting as well as the intensity and extent of β₂AR signalling from the cell surface.     

The HECT Domain of the Ubiquitin Ligase Rsp5 Contributes to Substrate Recognition

Ubiquitin modification of endosomal membrane proteins is a signal for active inclusion into the Multivesicular Body (MVB) pathway, resulting in lysosomal degradation. However, the endosome represents a dynamic site of protein sorting with a majority of proteins destined for recycling, rather than MVB targeting. Substrate recognition by ubiquitin ligases is therefore highly regulated. We have investigated substrate recognition by the Nedd4 ortholog Rsp5 as a model for understanding ligase-substrate interactions. Rsp5 interacts directly with its substrate Cps1 via a novel interaction mode. Perturbation of this mode of interaction revealed a compensatory role for the Rsp5 adaptor Bsd2. These results highlight the ability of Rsp5 to interact with substrates via multiple modalities, suggesting additional mechanisms of regulating this interaction and relevant outcomes.     

Recycling of the epidermal growth factor receptor is mediated by a novel form of the clathrin adaptor protein Eps15

Levels of the epidermal growth factor receptor (EGFR) at the cell surface are tightly regulated by a complex endocytic machinery. Following internalization, EGFR is either recycled back to the cell surface or transported to the late endosome/lysosome for degradation. Currently, the molecular machinery that regulates this sorting pathway is only partially defined. Eps15 (EGFR pathway substrate 15) is an endocytic adaptor protein that is well known to support clathrin-mediated internalization of EGFR at the plasma membrane. Using RT-PCR, we have identified a novel short form of Eps15 (Eps15S) from rat liver that lacks the 111 C-terminal amino acids present in the traditional Eps15 form. The goal of this study was to define the functional role of the novel Eps15S form in EGFR trafficking. Overexpression of a mutant form of Eps15S (Eps15S ΔEH2/EH3) did not block EGFR internalization but reduced its recycling to the cell surface. After knockdown of all Eps15 forms, re-expression of Eps15S significantly reduced EGFR degradation while promoting recycling back to the cell surface. In contrast, re-expression of Eps15 did not potentiate receptor recycling. Furthermore, overexpression of the mutant Eps15S substantially reduced cell proliferation, linking EGFR recycling to downstream mitogenic effects. Finally, we found that Eps15S is localized to the Rab11-positive recycling endosome that is disrupted in cells expressing the Eps15S mutant, leading to an accumulation of the EGFR in early endosomes. These findings suggest that distinct forms of Eps15 direct EGFR to either the late endosome/lysosome for degradation (Eps15) or to the recycling endosome for transit back to the cell surface (Eps15S).     

A tyrosine-based sorting signal regulates intracellular trafficking of protease-activated receptor-1: multiple regulatory mechanisms for agonist-induced G protein-coupled receptor internalization

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly proteolytically activated, internalized, and then sorted to lysosomes and degraded. Internalization and lysosomal sorting of activated PAR1 is critical for termination of receptor signaling. We previously demonstrated that activated PAR1 is rapidly phosphorylated and internalized via a clathrin- and dynamin-dependent pathway that is independent of arrestins. Toward understanding the mechanisms responsible for activated PAR1 internalization through clathrin-coated pits we examined the function of a highly conserved tyrosine-based motif, YXXL, localized in the cytoplasmic carboxyl tail of the receptor. A mutant PAR1 in which tyrosine 383 and leucine 386 were replaced with alanines (Y383A/L386A) was significantly impaired in agonist-triggered internalization and degradation compared with wild-type receptor. In contrast, constitutive internalization, and recycling of unactivated PAR1 Y383A/L386A mutant was not affected, suggesting that tonic cycling of the mutant receptor remained intact. Strikingly, a PAR1 C387Z truncation mutant in which the YXXL motif was exposed at the C terminus constitutively internalized and degraded in an agonist-independent manner, whereas C387Z truncation mutant in which the critical tyrosine and leucine were mutated to alanine (C387Z-Y383A/L386A) failed to internalize. Inhibition of PAR1 C387Z mutant constitutive internalization with dominant-negative K44A dynamin blocked agonist-independent degradation of the mutant receptor. Together these findings strongly suggest that internalization of activated PAR1 is controlled by multiple regulatory mechanisms involving phosphorylation and a highly conserved tyrosine-based motif, YXXL. This study is the first to describe a function for a tyrosine-based motif, YXX, in GPCR internalization and reveal novel complexities in the regulation of GPCR trafficking.     

Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.     

Sorting Out the Sorting Functions of Endosomes in Arabidopsis

In animals, sorting of membrane proteins following their internalization from the plasma membrane (PM) by endocytosis occurs through a series of different endosomal compartments. In plants, how and where these sorting events take place is still poorly understood and our current view of the endocytic pathway still largely relies on analogies made from the animal system. However, extensive differences seem to exist between animal and plant endosomal functions, as exemplified by the role of the trans-Golgi network (TGN) as an early endosomal compartment in plants or the functional diversification of conserved sorting complexes. By using the Arabidopsis root tip as a reference model, we and other have begun to shed light on the complexity of the plant endocytic pathways. Notably, we have recently characterized the functions of an endosomal compartment, the SNX1-endosomes, also referred to as the prevacuolar compartment (PVC) or multivesicular bodies (MVB), in the sorting of different cargo proteins, including two related auxin-efflux carriers, PIN1 and PIN2. We have shown that routing decisions take place at this endosomal level, such as the sorting of PIN2 toward the lytic vacuole for degradation or PIN1 toward the PM for recycling.Key Words: Arabidopsis, intracellular trafficking, endocytic recycling, endosomes, MVB, PVC, VPS29, SNX, PIN, cell polarity     

Evidence for a direct role of the Doa4 deubiquitinating enzyme in protein sorting into the MVB pathway

Degradation of various membrane proteins in the lumen of the vacuole/lysosome requires their prior sorting into the multivesicular body (MVB) pathway. In this process, ubiquitin serves as a sorting signal for most cargoes. The yeast ubiquitin hydrolase Doa4 acts late in the MVB pathway. It's role is to catalyze deubiquitination of cargo proteins prior to their sorting into the endosomal vesicles. This step rescues ubiquitin from degradation in the vacuole/lysosome, enabling it to be recycled. Accordingly, the level of monomeric ubiquitin is typically reduced in doa4 mutants. Although MVB sorting of cargo proteins is also impaired in doa4 mutants, the question of whether this defect is due solely to Doa4's role in maintaining a normal pool of ubiquitin in the cell remains open. We here show that the requirement of Doa4 for correct MVB sorting of the endocytic cargo general amino acid permease and of the biosynthetic cargo carboxypeptidase S are not because of the role of Doa4 in ubiquitin recycling. This suggests a direct role of Doa4 in MVB sorting and we show that this role depends on Doa4's catalytic activity. We propose that deubiquitination by Doa4 of cargo proteins and/or some components of the MVB sorting machinery is essential to correct sorting of cargoes into the MVB pathway.