Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies

Doa4 is a ubiquitin-specific protease in Saccharomyces cerevisiae that deubiquitinates integral membrane proteins sorted into the lumenal vesicles of late-endosomal multivesicular bodies (MVBs). We show that the non-catalytic N terminus of Doa4 mediates its recruitment to endosomes through its association with Bro1, which is one of several highly conserved class E Vps proteins that comprise the core MVB sorting machinery. In turn, Bro1 directly stimulates deubiquitination by interacting with a YPxL motif in the catalytic domain of Doa4. Mutations in either Doa4 or Bro1 that disrupt catalytic activation of Doa4 impair deubiquitination and sorting of MVB cargo proteins and lead to the formation of lumenal MVB vesicles that are predominantly small compared with the vesicles seen in wild-type cells. Thus, by recruiting Doa4 to late endosomes and stimulating its catalytic activity, Bro1 fulfills a novel dual role in coordinating deubiquitination in the MVB pathway.     

Tetraspan cargo adaptors usher GPI-anchored proteins into multivesicular bodies

Ubiquitinated membrane proteins are sorted into intralumenal endosomal vesicles on their way for degradation in lysosomes. Here we summarize the discovery of the Cos proteins, which work to organize and segregate ubiquitinated cargo prior to its incorporation into intralumenal vesicles of the multivesicular body (MVB). Importantly, cargoes such as GPI-anchored proteins (GPI-APs) that cannot undergo ubiquitination, rely entirely on Cos proteins for sorting into intralumenal vesicles using the same pathway that depends on ESCRTs and ubiquitin ligases that typical polytopic membrane proteins do. Here we show Cos proteins provide functions as not only adaptor proteins for ubiquitin ligases, but also as cargo carriers that can physically usher a variety of other proteins into the MVB pathway. We then discuss the significance of this new sorting model and the broader implications for this cargo adaptor mechanism, whereby yeast Cos proteins, and their likely animal analogs, provide a ubiquitin sorting signal in trans to enable sorting of a membrane protein network into intralumenal vesicles.     

Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

ABSTRACT: BACKGROUND: In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. RESULTS: Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. CONCLUSIONS: Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.     

Molecular mechanisms of late endosome morphology, identity and sorting

Recent studies using electron microscopy, protein crystallography, classic biochemistry and novel live-cell imaging have provided numerous insights into the endocytic pathway, describing a dynamic system in which compartment morphology, molecular identity and the mechanics of cargo sorting are intimately connected. Current evidence supports a model of maturation in which the lipids, cargo proteins and Rab population at the endosome determine its competence to perform the functions of late endosomes, including the sorting of cargoes into lumenal vesicles and fusion with lysosomes.     

The Vps27p Hse1p complex binds ubiquitin and mediates endosomal protein sorting

Membrane proteins that are degraded in the vacuole of Saccharomyces cerevisiae are sorted into discrete intralumenal vesicles, analogous to the internal membranes of multi-vesiculated bodies (MVBs). Recently, it has shown that the attachment of ubiquitin (Ub) mediates sorting into lumenal membranes. We describe a complex of Vps27p and Hse1p that localizes to endosomal compartments and is required for the recycling of Golgi proteins, formation of lumenal membranes and sorting of ubiquitinated proteins into those membranes. The Vps27p-Hse1p complex binds to Ub and requires multiple Ub Interaction Motifs (UIMs). Mutation of these motifs results in specific defects in the sorting of ubiquitinated proteins into the vacuolar lumen. However, the recycling of Golgi proteins and the generation of lumenal membranes proceeds normally in Delta UIM mutants. These data support a model in which the Vps27p-Hse1p complex has multiple functions at the endosome, one of which is as a sorting receptor for ubiquitinated membrane proteins destined for degradation.     

Membrane dynamics and the biogenesis of lysosomes (Review)

Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca 2+ concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.     

Membrane dynamics and the biogenesis of lysosomes

Lysosomes are dynamic organelles receiving membrane traffic input from the biosynthetic, endocytic and autophagic pathways. They may be regarded as storage organelles for acid hydrolases and are capable of fusing with late endosomes to form hybrid organelles where digestion of endocytosed macromolecules occurs. Reformation of lysosomes from the hybrid organelles involves content condensation and probably removal of some membrane proteins by vesicular traffic. Lysosomes can also fuse with the plasma membrane in response to cell surface damage and a rise in cytosolic Ca(2+) concentration. This process is important in plasma membrane repair. The molecular basis of membrane traffic pathways involving lysosomes is increasingly understood, in large part because of the identification of many proteins required for protein traffic to vacuoles in the yeast Saccharomyces cerevisiae. Mammalian orthologues of these proteins have been identified and studied in the processes of vesicular delivery of newly synthesized lysosomal proteins from the trans-Golgi network, fusion of lysosomes with late endosomes and sorting of membrane proteins into lumenal vesicles. Several multi-protein oligomeric complexes required for these processes have been identified. The present review focuses on current understanding of the molecular mechanisms of fusion of lysosomes with both endosomes and the plasma membrane and on the sorting events required for delivery of newly synthesized membrane proteins, endocytosed membrane proteins and other endocytosed macromolecules to lysosomes.     

Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes

The sorting of transmembrane cargo proteins into the lumenal vesicles of multivesicular bodies (MVBs) depends on the recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomal membranes. The subsequent dissociation of ESCRT complexes from endosomes requires Vps4, a member of the AAA family of adenosine triphosphatases. We show that Did2 directs Vps4 activity to the dissociation of ESCRT-III but has no role in the dissociation of ESCRT-I or -II. Surprisingly, vesicle budding into the endosome lumen occurs in the absence of Did2 function even though Did2 is required for the efficient sorting of MVB cargo proteins into lumenal vesicles. This uncoupling of MVB cargo sorting and lumenal vesicle formation suggests that the Vps4-mediated dissociation of ESCRT-III is an essential step in the sorting of cargo proteins into MVB vesicles but is not a prerequisite for the budding of vesicles into the endosome lumen.     

Multivesicular bodies: co-ordinated progression to maturity

Multivesicular endosomes/bodies (MVBs) sort endocytosed proteins to different destinations. Many lysosomally directed membrane proteins are sorted onto intralumenal vesicles, whilst recycling proteins remain on the perimeter membrane from where they are removed via tubular extensions. MVBs move to the cell centre during this maturation process and, when all recycling proteins have been removed, fuse with lysosomes. Recent advances have identified endosomal-sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways in intralumenal vesicle formation and mechanisms for sorting recycling cargo into tubules. Cytoskeletal motors, through interactions with these machineries and by regulating MVB movement, help to co-ordinate events leading to a mature, fusion-competent MVB.     

Hrs and endocytic sorting of ubiquitinated membrane proteins

Endocytosed receptors are either recycled to the plasma membrane or trapped within intralumenal vesicles of multi-vesicular bodies for subsequent degradation in lysosomes. How the cell is able to sort receptors in endosomes has so far been largely unknown. The hepatocyte growth factor regulated tyrosine kinase substrate, Hrs, is an essential protein that has been implicated in cell signalling and intracellular membrane trafficking. Very recently, several reports have demonstrated a role for Hrs in endocytic sorting of ubiquitinated membrane proteins. Here, we review current knowledge about how Hrs recognises ubiquitinated cargo that is destined for lysosomal degradation, and how Hrs may act as a key regulator of the molecular machinery involved in receptor sorting and multivesicular body formation.     

Sna3 Is an Rsp5 Adaptor Protein that Relies on Ubiquitination for Its MVB Sorting

The process in which ubiquitin ( Ub ) conjugation is required for trafficking of integral membrane proteins into multivesicular bodies ( MVBs ) and eventual degradation in the lumen of lysosomes/vacuoles is well defined. However , Ub ‐independent pathways into MVBs are less understood. To better understand this process, we have further characterized the membrane protein Sna 3, the prototypical Ub ‐independent cargo protein sorted through the MVB pathway in yeast. We show that Sna 3 trafficking to the vacuole is critically dependent on Rsp 5 ligase activity and ubiquitination. We find Sna 3 undergoes Ub ‐dependent MVB sorting by either becoming ubiquitinated itself or associating with other ubiquitinated membrane protein substrates. In addition, our functional studies support a role for Sna 3 as an adaptor protein that recruits Rsp 5 to cargo such as the methionine transporter Mup 1, resulting in efficient Mup 1 delivery to the vacuole .     

DOA1/UFD3 plays a role in sorting ubiquitinated membrane proteins into multivesicular bodies

Ubiquitin (Ub) is a sorting signal that targets integral membrane proteins to the interior of the vacuole/lysosome by directing them into lumenal vesicles of multivesicular bodies (MVBs). The Vps27-Hse1 complex, which is homologous to the Hrs-STAM complex in mammalian cells, serves as a Ub-sorting receptor at the surface of early endosomes. We have found that Hse1 interacts with Doa1/Ufd3. Doa1 is known to interact with Cdc48/p97 and Ub and is required for maintaining Ub levels. We find that the Hse1 Src homology 3 domain binds directly to the central PFU domain of Doa1. Mutations in Doa1 that block Hse1 binding but not Ub binding do not alter Ub levels but do result in the missorting of the MVB cargo GFP-Cps1. Loss of Doa1 also causes a synthetic growth defect when combined with loss of Vps27. Unlike the loss of Doa1 alone, the doa1Delta vps27Delta double mutant phenotype is not suppressed by Ub overexpression, demonstrating that the effect is not due to indirect consequence of lowered Ub levels. Loss of Doa1 results in a defect in the accumulation of GFP-Ub within yeast vacuoles, implying that there is a reduction in the flux of ubiquitinated membrane proteins through the MVB pathway. This defect was also reflected by an inability to properly sort Vph1-GFP-Ub, a modified subunit of the multiprotein vacuolar ATPase complex, which carries an in-frame fusion of Ub as an MVB sorting signal. These results reveal novel roles for Doa1 in helping to process ubiquitinated membrane proteins for sorting into MVBs.     

Intra-endosomal membrane traffic

Following endocytosis, ubiquitinated signaling receptors are incorporated within intraluminal vesicles of forming multivesicular endosomes. These vesicles then follow the pathway from early to late endosomes, remaining within the endosomal lumen, and are eventually delivered to lysosomes, where they are degraded together with their protein cargo. However, intraluminal vesicles do not always end up in lysosomes for degradation; they can also fuse back with the limiting membrane of late endosomes. This route, which might be regulated by lyso-bisphosphatidic acid and its putative effector Alix, can be hijacked by the anthrax toxin and vesicular stomatitis virus and is presumably exploited by proteins and lipids that transit through intraluminal vesicles. Alternatively, these vesicles can be released extracellularly, like HIV in macrophages, upon fusion of endosomes or lysosomes with the plasma membrane.     

Regulators of Vps4 ATPase Activity at Endosomes Differentially Influence the Size and Rate of Formation of Intralumenal Vesicles

Recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomes regulates selective inclusion of transmembrane proteins into the lumenal vesicles of multivesicular bodies (MVBs). ESCRT-0, -I, and -II bind directly to ubiquitinated transmembrane cargoes of the MVB pathway, whereas polymerization of ESCRT-III at endosomes is thought to bend the membrane and/or provide the energetic force that drives membrane scission and detachment of vesicles into the endosome lumen. Disassembly of the ESCRT-III polymer and dissociation of its subunits from endosomes requires the Vps4 ATPase, the activity of which is controlled in vivo by regulatory proteins. We identify distinct spatiotemporal roles for Vps4-regulating proteins through examinations of subcellular localization and endosome morphology. Did2 plays a unique role in the regulation of MVB lumenal vesicle size, whereas Vtal and Vps60 promote efficient membrane scission and delivery of membrane to the endosome lumen. These morphological effects probably result from Vps4-mediated manipulations of ESCRT-III, because we show dissociation of ESCRT-0, -I, and -II from endosomes is not directly dependent on Vps4 activity.     

Toxin pores endocytosed during plasma membrane repair traffic into the lumen of MVBs for degradation

Cells permeabilized by the bacterial pore-forming toxin streptolysin O (SLO) reseal their plasma membrane in a Ca(2+) -dependent manner. Resealing involves Ca(2+) -dependent exocytosis of lysosomes, release of acid sphingomyelinase and rapid formation of endosomes that carry the transmembrane pores into the cell. The intracellular fate of the toxin-carrying endocytic vesicles, however, is still unknown. Here, we show that SLO pores removed from the plasma membrane by endocytosis are sorted into the lumen of lysosomes, where they are degraded. SLO-permeabilized cells contain elevated numbers of total endosomes, which increase gradually in size while transitioning from endosomes with flat clathrin coats to large multivesicular bodies (MVBs). Under conditions that allow endocytosis and plasma membrane repair, SLO is rapidly ubiquitinated and gradually degraded, in a process sensitive to inhibitors of lysosomal hydrolysis but not of proteasomes. The endosomes induced by SLO permeabilization become increasingly acidified and promote SLO degradation under normal conditions, but not in cells silenced for expression of Vps24, an ESCRT-III complex component required for the release of intraluminal vesicles into MVBs. Thus, cells dispose of SLO transmembrane pores by ubiquitination/ESCRT-dependent sorting into the lumen of late endosomes/lysosomes.     

Vps4 regulates a subset of protein interactions at the multivesicular endosome

During endocytic transport, specific integral membrane proteins are sorted into intraluminal vesicles that bud from the limiting membrane of the endosome. This process, known as multivesicular body (MVB) sorting, is important for several important biological processes. Moreover, components of the MVB sorting machinery are implicated in virus budding. During MVB sorting, a cargo protein recruits components of the MVB sorting machinery from cytoplasmic pools and these sequentially assemble on the endosome. Disassembly of these proteins and recycling into the cytoplasm is critical for MVB sorting. Vacuolar protein sorting 4 (Vps4) is an AAA (ATPase associated with a variety of cellular activities) ATPase which has been proposed to play a critical role in disassembly of the MVB sorting machinery. However, the mechanism by which it disassembles the complex is not clear. Vps4 contains an N-terminal microtubule interacting and trafficking (MIT) domain, which has previously been shown to be required for recruitment to endosomes, and a single AAA ATPase domain, the activity of which is required for Vps4 function. In this study we have systematically characterized the interaction of Vps4 with other components of the MVB sorting machinery. We demonstrate that Vps4 interacts directly with Vps2 and Bro1. We also show that a subset of Vps4 interactions is regulated by ATP hydrolysis, and one interaction is regulated by ATP binding. Finally, we show that most proteins interact with the Vps4 MIT domain. Our studies indicate that the MIT domain has a dual role in substrate binding and recruitment to endosomes and indicate that Vps4 disassembles the MVB sorting machinery by direct effects on multiple proteins.     

Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain

Ubiquitination serves as a key sorting signal in the lysosomal degradation of endocytosed receptors through the ability of ubiquitinated membrane proteins to be recognized and sorted by ubiquitin-binding proteins along the endocytic route. The ESCRT-II complex in yeast contains one such protein, Vps36, which harbors a ubiquitin-binding NZF domain and is required for vacuolar sorting of ubiquitinated membrane proteins. Surprisingly, the presumptive mammalian ortholog Eap45 lacks the ubiquitin-binding module of Vps36, and it is thus not clear whether mammalian ESCRT-II functions to bind ubiquitinated cargo. In this paper, we provide evidence that Eap45 contains a novel ubiquitin-binding domain, GLUE (GRAM-like ubiquitin-binding in Eap45), which binds ubiquitin with similar affinity and specificity as other ubiquitin-binding domains. The GLUE domain shares similarities in its primary and predicted secondary structures to phosphoinositide-binding GRAM and PH domains. Accordingly, we find that Eap45 binds to a subset of 3-phosphoinositides, suggesting that ubiquitin recognition could be coordinated with phosphoinositide binding. Furthermore, we show that Eap45 colocalizes with ubiquitinated proteins on late endosomes. These results are consistent with a role for Eap45 in endosomal sorting of ubiquitinated cargo.     

Hrs and SNX3 functions in sorting and membrane invagination within multivesicular bodies

After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs—an adaptor-like protein that binds membrane PtdIns3P via a FYVE motif—and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.     

ESCRT proteins and cell signalling

Subunits of the endosomal sorting complex required for transport (ESCRT) were identified as components of a molecular machinery that sorts ubiquitinated membrane proteins into the intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) for subsequent delivery to the lumen of lysosomes or related organelles. As many of the membrane proteins that undergo ESCRT-mediated sorting are signalling receptors that are ubiquitinated in response to ligand binding, ESCRT subunits have been hypothesized to play a crucial role in attenuation of cell signalling by mediating ligand-induced receptor degradation. Here we discuss this concept based on the examples from loss-of-function studies in model organisms and cell lines. The emerging picture is that ESCRTs are indeed involved in downregulation of receptor signalling pathways associated with cell survival, proliferation and polarity. In addition, the recent discovery of a positive role for the ESCRT pathway in Wnt signalling through sequestration of an inhibitory cytosolic component into MVEs illustrates that ESCRTs may also control signalling in ways that are independent of degradative receptor sorting.     

Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes

After endocytosis, some membrane proteins recycle from early endosomes to the plasma membrane whereas others are transported to late endosomes and lysosomes for degradation. Conjugation with the small polypeptide ubiquitin is a signal for lysosomal sorting. Here we show that the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, is involved in the endosomal sorting of ubiquitinated membrane proteins. Hrs contains a clathrin-binding domain, and by electron microscopy we show that Hrs localizes to flat clathrin lattices on early endosomes. We demonstrate that Hrs binds directly to ubiquitin by way of a ubiquitin-interacting motif (UIM), and that ubiquitinated proteins localize specifically to Hrs- and clathrin-containing microdomains. Whereas endocytosed transferrin receptors fail to colocalize with Hrs and rapidly recycle to the cell surface, transferrin receptors that are fused to ubiquitin interact with Hrs, localize to Hrs- and clathrin-containing microdomains and are sorted to the degradative pathway. Overexpression of Hrs strongly and specifically inhibits recycling of ubiquitinated transferrin receptors by a mechanism that requires a functional UIM. We conclude that Hrs sorts ubiquitinated membrane proteins into clathrin-coated microdomains of early endosomes, thereby preventing their recycling to the cell surface.