Vps9p CUE domain ubiquitin binding is required for efficient endocytic protein traffic

Rab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway.     

Ubiquitin binding by the CUE domain promotes endosomal localization of the Rab5 GEF Vps9

Vps9 and Muk1 are guanine nucleotide exchange factors (GEFs) in Saccharomyces cerevisiae that regulate membrane trafficking in the endolysosomal pathway by activating Rab5 GTPases. We show that Vps9 is the primary Rab5 GEF required for biogenesis of late endosomal multivesicular bodies (MVBs). However, only Vps9 (but not Muk1) is required for the formation of aberrant class E compartments that arise upon dysfunction of endosomal sorting complexes required for transport (ESCRTs). ESCRT dysfunction causes ubiquitinated transmembrane proteins to accumulate at endosomes, and we demonstrate that endosomal recruitment of Vps9 is promoted by its ubiquitin-binding CUE domain. Muk1 lacks ubiquitin-binding motifs, but its fusion to the Vps9 CUE domain allows Muk1 to rescue endosome morphology, cargo trafficking, and cellular stress-tolerance phenotypes that result from loss of Vps9 function. These results indicate that ubiquitin binding by the CUE domain promotes Vps9 function in endolysosomal membrane trafficking via promotion of localization.     

CUE domain-containing protein Vps901 is required for vacuolar protein transport in Schizosaccharomyces pombe

The functions of two Schizosaccharomyces pombe Vps9-like genes, SPBC4F6.10/vps901(+) and SPBC29A10.11c/vps902(+), were characterized. Genomic sequence analysis predicted that Vps901p contains a VPS9 domain, whereas cDNA analyses revealed that Vps901p contains a CUE domain (coupling of ubiquitin to ER degradation) in its C-terminal region. Deletion of vps901(+) resulted in mis-sorting and secretion of S. pombe vacuolar carboxypeptidase Cpy1p, whereas deletion of vps902(+) had no effect, suggesting that only Vps901p functions in vacuolar protein transport in S. pombe. Deletion of vps901(+) further produced pleiotropic phenotypes, including vacuolar homotypic fusion and endocytosis defects. Heterologous expression of the budding yeast VPS9 gene corrected the CPY mis-sorting defect in vps901Δ cells. These findings suggest that the VPS9 domain of Vps901p is required for vacuolar protein trafficking in S. pombe.     

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.     

Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases

VPS10 (Vacuolar Protein Sorting) encodes a large type I transmembrane protein (Vps10p), involved in the sorting of the soluble vacuolar hydrolase carboxypeptidase Y (CPY) to the Saccharomyces cerevisiae lysosome-like vacuole. Cells lacking Vps10p missorted greater than 90% CPY and 50% of another vacuolar hydrolase, PrA, to the cell surface. In vitro equilibrium binding studies established that the 1,380-amino acid lumenal domain of Vps10p binds CPY precursor in a 1:1 stoichiometry, further supporting the assignment of Vps10p as the CPY sorting receptor. Vps10p has been immunolocalized to the late-Golgi compartment where CPY is sorted away from the secretory pathway. Vps10p is synthesized at a rate 20-fold lower that that of its ligand CPY, which in light of the 1:1 binding stoichiometry, requires that Vps10p must recycle and perform multiple rounds of CPY sorting. The 164-amino acid Vps10p cytosolic domain is involved in receptor trafficking, as deletion of this domain resulted in delivery of the mutant Vps10p to the vacuole, the default destination for membrane proteins in yeast. A tyrosine-based signal (YSSL80) within the cytosolic domain enables Vps10p to cycle between the late-Golgi and prevacuolar/endosomal compartments. This tyrosine-based signal is homologous to the recycling signal of the mammalian mannose-6-phosphate receptor. A second yeast gene, VTH2, encodes a protein highly homologous to Vps10p which, when over-produced, is capable of suppressing the CPY and PrA missorting defects of a vps10 delta strain. These results indicate that a family of related receptors act to target soluble hydrolases to the vacuole.     

Mechanism of ubiquitin recognition by the CUE domain of Vps9p

Coupling of ubiquitin conjugation to ER degradation (CUE) domains are approximately 50 amino acid monoubiquitin binding motifs found in proteins of trafficking and ubiquitination pathways. The 2.3 A structure of the Vps9p-CUE domain is a dimeric domain-swapped variant of the ubiquitin binding UBA domain. The 1.7 A structure of the CUE:ubiquitin complex shows that one CUE dimer binds one ubiquitin molecule. The bound CUE dimer is kinked relative to the unbound CUE dimer and wraps around ubiquitin. The CUE monomer contains two ubiquitin binding surfaces on opposite faces of the molecule that cannot bind simultaneously to a single ubiquitin molecule. Dimerization of the CUE domain allows both surfaces to contact a single ubiquitin molecule, providing a mechanism for high-affinity binding to monoubiquitin.     

Demonstration in yeast of the function of BP-80, a putative plant vacuolar sorting receptor

BP-80, later renamed VSR(PS-1), is a putative receptor involved in sorting proteins such as proaleurain to the lytic vacuole, with its N-terminal domain recognizing the vacuolar sorting determinant. Although all VSR(PS-1) characteristics and in vitro binding properties described so far favored its receptor function, this function remained to be demonstrated. Here, we used green fluorescent protein (GFP) as a reporter in a yeast mutant strain defective for its own vacuolar receptor, Vps10p. By expressing VSR(PS-1) together with GFP fused to the vacuolar sorting determinant of petunia proaleurain, we were able to efficiently redirect the reporter to the yeast vacuole. VSR(PS-1) is ineffective on GFP either alone or when fused with another type of plant vacuolar sorting determinant from a chitinase. The plant VSR(PS-1) therefore interacts specifically with the proaleurain vacuolar sorting determinant in vivo, and this interaction leads to the transport of the reporter protein through the yeast secretory pathway to the vacuole. This finding demonstrates VSR(PS-1) receptor function but also emphasizes the differences in the spectrum of ligands between Vps10p and its plant equivalent.     

A ubiquitin-binding motif required for intramolecular monoubiquitylation,the CUE domain

Monoubiquitylation is a regulatory signal, like phosphorylation, that can alter the activity, location or structure of a protein. Monoubiquitin signals are likely to be recognized by ubiquitin-binding proteins that transmit the regulatory information conferred by monoubiquitylation. To identify monoubiquitin-binding proteins, we used a mutant ubiquitin that lacks the primary site of polyubiquitin chain formation as bait in a two-hybrid screen. The C-terminus of Vps9, a protein required in the yeast endocytic pathway, interacted specifically with monoubiquitin. The region required for monoubiquitin binding mapped to the Vps9 CUE domain, a sequence previously identified by database searches as similar to parts of the yeast Cue1 and mammalian Tollip proteins. We demonstrate that CUE domains bind directly to monoubiquitin and we have defined crucial interaction surfaces on both binding partners. The Vps9 CUE domain is required to promote monoubiquitylation of Vps9 by the Rsp5 hect domain ubiquitin ligase. Thus, we conclude that the CUE motif is an evolutionarily conserved monoubiquitin-binding domain that mediates intramolecular monoubiquitylation.     

A yeast protein related to a mammalian Ras-binding protein, Vps9p, is required for localization of vacuolar proteins.

In the yeast Saccharomyces cerevisiae, mutations in vacuolar protein sorting (VPS) genes result in secretion of proteins normally localized to the vacuole. Characterization of the VPS pathway has provided considerable insight into mechanisms of protein sorting and vesicle-mediated intracellular transport. We have cloned VPS9 by complementation of the vacuolar protein sorting defect of vps9 cells, characterized its gene product, and investigated its role in vacuolar protein sorting. Cells with a vps9 disruption exhibit severe vacuolar protein sorting defects and a temperature-sensitive growth defect at 38 degrees C. Electron microscopic examination of delta vps9 cells revealed the appearance of novel reticular membrane structures as well as an accumulation of 40- to 50-nm-diameter vesicles, suggesting that Vps9p may be required for the consumption of transport vesicles containing vacuolar protein precursors. A temperature-conditional allele of vps9 was constructed and used to investigate the function of Vps9p. Immediately upon shifting of temperature-conditional vps9 cells to the nonpermissive temperature, newly synthesized carboxypeptidase Y was secreted, indicating that Vps9p function is directly required in the VPS pathway. Antibodies raised against Vps9p immunoprecipitate a rare 52-kDa protein that fractionates with cytosolic proteins following cell lysis and centrifugation. Analysis of the VPS9 DNA sequence predicts that Vps9p is related to human proteins that bind Ras and negatively regulate Ras-mediated signaling. We term the related regions of Vps9p and these Ras-binding proteins a GTPase binding homology domain and suggest that it defines a family of proteins that bind monomeric GTPases. Vps9p may bind and serve as an effector of a rab GTPase, like Vps2lp, required for vacuolar protein sorting.     

Complicated trafficking behaviors involved in paradoxical regulation of sortilin in lipid metabolism

This review aims to summarize and discuss the most recent advances in our understanding of the underlying mechanisms of the paradoxical effects of sortilin on lipid metabolism. The vacuolar protein sorting 10 protein (Vps10p) domain in the sortilin protein is responsible for substrate binding. Its cytoplasmic tail interacts with adaptor molecules, and modifications can determine whether sortilin trafficking occurs via the anterograde or retrograde pathway. The complicated trafficking behaviors likely contribute to the paradoxical roles of sortilin in lipid metabolism. The anterograde pathway of sortilin trafficking in hepatocytes, enterocytes, and peripheral cells likely causes an increase in plasma lipid levels, while the retrograde pathway leads to the opposite effect. Hepatocyte sortilin functions via the anterograde or retrograde pathway in a complicated and paradoxical manner to regulate apoB-containing lipoprotein metabolism. Clarifying the regulatory mechanisms underlying the trafficking behaviors of sortilin is necessary and may lead to artificial sortilin intervention as a potential therapeutic strategy for lipid disorder diseases. Conclusively, the paradoxical regulation of sortilin in lipid metabolism is likely due to its complicated trafficking behaviors.     

Vps10p transport from the trans-Golgi network to the endosome is mediated by clathrin-coated vesicles

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.     

Vps27-Hse1 and ESCRT-I complexes cooperate to increase efficiency of sorting ubiquitinated proteins at the endosome

Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein complexes have been proposed as Ub-sorting "receptors," the Vps27-Hse1 complex and the ESCRT-I complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.     

Vps41p function in the alkaline phosphatase pathway requires homo-oligomerization and interaction with AP-3 through two distinct domains

Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein-dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.     

Plant RMR proteins: unique vacuolar sorting receptors that couple ligand sorting with membrane internalization

In receptor-mediated sorting of soluble protein ligands in the endomembrane system of eukaryotic cells, three completely different receptor proteins for mammalian (mannose 6-phosphate receptor), yeast (Vps10p) and plant cells (vacuolar sorting receptor; VSR) have in common the features of pH-dependent ligand binding and receptor recycling. In striking contrast, the plant receptor homology-transmembrane-RING-H2 (RMR) proteins serve as sorting receptors to a separate type of vacuole, the protein storage vacuole, but do not recycle, and their trafficking pathway results in their internalization into the destination vacuole. Even though plant RMR proteins share high sequence similarity with the best-characterized mammalian PA-TM-RING family proteins, these two families of proteins appear to play distinctly different roles in plant and animal cells. Thus, this minireview focuses on this unique sorting mechanism and traffic of RMR proteins via dense vesicles in various plant cell types.     

Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae.

Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand binding to Vps10p by introducing deletions in the lumenal region. This region contains two domains with homology to each other. Domain 2 binds carboxypeptidase Y (CPY), proteinase A (PrA) and hybrids of these proteases with invertase. Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p. The native proteases compete for binding to domain 2. Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p-dependent sorting of CPY-invertase. Apparently, domain 2 contains two different binding sites; one for APY, CPY and PrA, and one for CPY-invertase and PrA-invertase. The latter interaction seems not to be sequence specific, and we suggest that an unfolded structure in these ligands is recognized by Vps10p.     

Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a mammalian homologue of yeast vacuolar protein sorting (Vps) protein Vps27p; however, the role of Hrs in lysosomal trafficking is unclear. Here, we report that Hrs interacts with sorting nexin 1 (SNX1), a recently identified mammalian homologue of yeast Vps5p that recognizes the lysosomal targeting code of epidermal growth factor receptor (EGFR) and participates in lysosomal trafficking of the receptor. Biochemical analyses demonstrate that Hrs and SNX1 are ubiquitous proteins that exist in both cytosolic and membrane-associated pools, and that the association of Hrs and SNX occurs on cellular membranes but not in the cytosol. Furthermore, endogenous SNX1 and Hrs form a approximately 550-kDa complex that excludes EGFR. Immunofluorescence and subcellular fractionation studies show that Hrs and SNX1 colocalize on early endosomes. By using deletion analysis, we have mapped the binding domains of Hrs and SNX1 that mediate their association. Overexpression of Hrs or its SNX1-binding domain inhibits ligand-induced degradation of EGFR, but does not affect either constitutive or ligand-induced receptor-mediated endocytosis. These results suggest that Hrs may regulate lysosomal trafficking through its interaction with SNX1.     

N-linked oligosaccharides on the low density lipoprotein receptor homolog SorLA/LR11 are modified with terminal GalNAc-4-SO4 in kidney and brain

Sorting protein-related receptor (SorLA/LR11) is a highly conserved mosaic receptor that is expressed by cells in a number of different tissues including principal cells of the collecting ducts in the kidney and neurons in the central and peripheral nervous systems. SorLA/LR11 has features that indicate it serves as a sorting receptor shuttling between the plasma membrane, endosomes, and the Golgi. We have found that a fraction of SorLA/LR11 that is synthesized in the kidney and the brain bears N-linked oligosaccharides that are modified with terminal beta1,4-linked GalNAc-4-SO(4). Oligosaccharides located in the vacuolar sorting (Vps) 10p domain (Vps10p domain) are modified with beta1,4-linked GalNAc when the Vps10p domain is expressed in cells along with either of two recently cloned protein-specific beta1,4GalNAc-transferases, GalNAcTIII and GalNAcTIV. Either of two sequences with basic amino acids located within the Vps10p domain is able to mediate recognition by these beta1,4GalNAc-transferases. The highly specific modification of oligosaccharides in the Vps10p domain of SorLA/LR11 with terminal GalNAc-4-SO(4) suggests that this unusual modification may modulate the interaction of SorLA/LR11 with proteins and influence their trafficking.     

PXA Domain-Containing Protein Pxa1 Is Required for Normal Vacuole Function and Morphology in Schizosaccharomyces pombe

PhoX homology (PX) domain-containing proteins play critical roles in vesicular trafficking, protein sorting, and lipid modification in eukaryotic cells. Several proteins with PX domains contain an associated domain termed PXA (PX-associated). Although PXA domain-containing proteins are required for some important cellular processes, the function of the PXA domain is unknown. We identified three PXA domain-containing proteins in Schizosaccharomyces pombe. S. pombe Pxa1p (SPAC5D6.07c) contained only the PXA domain, not the PX domain. To elucidate the role of the PXA domain in eukaryotic cells, we constructed and characterized a disruption mutant, pxa1. The pxa1 disruptant contained enlarged vacuoles and exhibited mislocalization of vacuolar carboxypeptidase Y (CPY). The conversion rate from pro- to mature-CPY was greatly impaired in pxa1 cells, and fluorescence microscopy indicated that a sorting receptor for CPY, Vps10p, mislocalized to the vacuolar membrane. The mutants were also deficient in vacuolar sorting of a multivesicular body (MVB) marker, a ubiquitin–GFP–carboxypeptidase S (Ub–GFP–CPS) fusion protein. Taken together, these results indicate that Pxa1 protein is required for normal vacuole function and morphology in S. pombe.     

Targeting of Sna3p to the endosomal pathway depends on its interaction with Rsp5p and multivesicular body sorting on its ubiquitylation

Rsp5p is an ubiquitin (Ub)-protein ligase of the Nedd4 family that carries WW domains involved in interaction with PPXY-containing proteins. It plays a key role at several stages of intracellular trafficking, such as Ub-mediated internalization of endocytic cargoes and Ub-mediated sorting of membrane proteins to internal vesicles of multivesicular bodies (MVBs), a process that is crucial for their subsequent targeting to the vacuolar lumen. Sna3p is a membrane protein previously described as an Ub-independent MVB cargo, but proteomic studies have since shown it to be an ubiquitylated protein. Sna3p carries a PPXY motif. We observed that this motif mediates its interaction with Rsp5p WW domains. Mutation of either the Sna3p PPXY motif or the Rsp5p WW3 domain or reduction in the amounts of Rsp5 results in the mistargeting of Sna3p to multiple mobile vesicles and prevents its sorting to the endosomal pathway. This sorting defect appears to occur prior to the defect displayed in rsp5 mutants by other MVB cargoes, which are correctly sorted to the endosomal pathway but missorted to the vacuolar membrane instead of the vacuolar lumen. Sna3p is polyubiquitylated on one target lysine, and a mutant Sna3p lacking its target lysine displays defective MVB sorting. Sna3p undergoes Rsp5-dependent polyubiquitylation, with K63-linked Ub chains.     

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.