MARCH-XI, a novel transmembrane ubiquitin ligase implicated in ubiquitin-dependent protein sorting in developing spermatids

A mechanism by which ubiquitinated cargo proteins are sorted into multivesicular bodies (MVBs) from plasma and trans-Golgi network (TGN) membranes is well established in yeast and mammalian somatic cells. However, the ubiquitin-dependent sorting pathway has not been clearly defined in germ cells. In this study we identified a novel member of the transmembrane RING-finger family of proteins, termed membrane-associated RING-CH (MARCH)-XI, that is expressed predominantly in developing spermatids and weakly in brain and pituitary. MARCH-XI possesses an E3 ubiquitin ligase activity that targets CD4 for ubiquitination. Immunoelectron microscopy of rat round spermatids showed that MARCH-XI is localized to TGN-derived vesicles and MVBs. Fluorescence staining of rat round spermatids and immunoprecipitation of rat testis demonstrated that MARCH-XI forms complexes with the adaptor protein complex-1 and with fucose-containing glycoproteins including ubiquitinated forms. Furthermore, the C-terminal region of MARCH-XI mediates its interaction with mu1-adaptin and Veli through a tyrosine-based motif and a PDZ binding motif, respectively. Our data suggest that MARCH-XI acts as a ubiquitin ligase with a role in ubiquitin-mediated protein sorting in the TGN-MVB transport pathway, which may be involved in mammalian spermiogenesis.     

Multivesicular body sorting: ubiquitin ligase Rsp5 is required for the modification and sorting of carboxypeptidase S

The multivesicular body (MVB) sorting pathway provides a mechanism for delivering transmembrane proteins into the lumen of the lysosome/vacuole. Recent studies demonstrated that ubiquitin modification acts in cis as a signal for the sorting of cargoes into this pathway. Here, we present results from a genetic selection designed to identify mutants that missort MVB cargoes. This selection identified a point mutation in ubiquitin ligase Rsp5 (Rsp5-326). At the permissive temperature, this mutant is specifically defective for ubiquitination and sorting of the ubiquitin-dependent MVB cargo precursor carboxypeptidase S (pCPS), but not ligand-induced ubiquitination of Ste2. A previous study implicated Tul1 as the ubiquitin ligase responsible for MVB sorting of pCPS. However, we detected no defect in either the sorting or ubiquitination of pCPS in tul1 mutants. We had previously shown that Fab1 phosphatidylinositol 3-phosphate 5-kinase is also required for MVB sorting of pCPS, but not Ste2. However, our analyses reveal that fab1 mutants do not exhibit a defect in ubiquitination of pCPS. Thus, both Rsp5 and Fab1 play distinct and essential roles in the targeting of biosynthetic MVB cargoes. However, whereas Rsp5 seems to be responsible for cargo ubiquitination, the precise role for Fab1 remains to be elucidated.     

MARCH-II is a syntaxin-6-binding protein involved in endosomal trafficking

Membrane-associated RING-CH (MARCH) is a recently identified member of the mammalian E3 ubiquitin ligase family, some members of which down-regulate the expression of immune recognition molecules. Here, we have identified MARCH-II, which is ubiquitously expressed and localized to endosomal vesicles and the plasma membrane. Immunoprecipitation and in vitro binding studies established that MARCH-II directly associates with syntaxin 6. Overexpression of MARCH-II resulted in redistribution of syntaxin 6 as well as some syntaxin-6-interacting soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into the MARCH-II-positive vesicles. In addition, the retrograde transport of TGN38 and a chimeric version of furin to trans-Golgi network (TGN) was perturbed--without affecting the endocytic degradative and biosynthetic secretory pathways--similar to effects caused by a syntaxin 6 mutant lacking the transmembrane domain. MARCH-II overexpression markedly reduced the cell surface expression of transferrin (Tf) receptor and Tf uptake and interfered with delivery of internalized Tf to perinuclear recycling endosomes. Depletion of MARCH-II by small interfering RNA perturbed the TGN localization of syntaxin 6 and TGN38/46. MARCH-II is thus likely a regulator of trafficking between the TGN and endosomes, which is a novel function for the MARCH family.     

DLG1 is an anchor for the E3 ligase MARCH2 at sites of cell-cell contact

PDZ domain containing molecular scaffolds plays a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination. Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination.     

A single ubiquitin is sufficient for cargo protein entry into MVBs in the absence of ESCRT ubiquitination

ESCRTs (endosomal sorting complexes required for transport) bind and sequester ubiquitinated membrane proteins and usher them into multivesicular bodies (MVBs). As Ubiquitin (Ub)-binding proteins, ESCRTs themselves become ubiquitinated. However, it is unclear whether this regulates a critical aspect of their function or is a nonspecific consequence of their association with the Ub system. We investigated whether ubiquitination of the ESCRTs was required for their ability to sort cargo into the MVB lumen. Although we found that Rsp5 was the main Ub ligase responsible for ubiquitination of ESCRT-0, elimination of Rsp5 or elimination of the ubiquitinatable lysines within ESCRT-0 did not affect MVB sorting. Moreover, by fusing the catalytic domain of deubiquitinating peptidases onto ESCRTs, we could block ESCRT ubiquitination and the sorting of proteins that undergo Rsp5-dependent ubiquitination. Yet, proteins fused to a single Ub moiety were efficiently delivered to the MVB lumen, which strongly indicates that a single Ub is sufficient in sorting MVBs in the absence of ESCRT ubiquitination.     

Characterization of multiple multivesicular body sorting determinants within Sna3: a role for the ubiquitin ligase Rsp5

A subset of proteins that transit the endosomal system are directed into the intralumenal vesicles of multivesicular bodies (MVBs). MVB formation is critical for a variety of cellular functions including receptor down-regulation, viral budding, antigen presentation, and the generation of lysosome-related organelles. Entry of transmembrane proteins into the intralumenal vesicles of a MVB is a highly regulated process that is positively modulated by covalent modification of cargoes with ubiquitin. To identify additional MVB sorting signals, we examined the previously described ubiquitination-independent MVB cargo Sna3. Although Sna3 ubiquitination is not essential, Sna3 MVB sorting is positively modulated by its ubiquitination. Examination of MVB sorting determinants within a form of Sna3 lacking all lysine residues identified two critical regions: an amino-terminal tyrosine-containing region and a carboxyl-terminal PPAY motif. This PPAY motif interacts with the WW domains of the ubiquitin ligase Rsp5, and mutations in either the WW or, surprisingly, the HECT domains of Rsp5 negatively impacted MVB targeting of lysine-minus Sna3. These data indicate that Rsp5 function is required for MVB targeting of Sna3 in a capacity beyond cargo ubiquitination. These results uncover a series of determinants impacting Sna3 MVB sorting, including unexpected roles for Rsp5.     

Hse1, a component of the yeast Hrs-STAM ubiquitin-sorting complex, associates with ubiquitin peptidases and a ligase to control sorting efficiency into multivesicular bodies

Ubiquitinated integral membrane proteins are delivered to the interior of the lysosome/vacuole for degradation. This process relies on specific ubiquitination of potential cargo and recognition of that Ub-cargo by sorting receptors at multiple compartments. We show that the endosomal Hse1-Vps27 sorting receptor binds to ubiquitin peptidases and the ubiquitin ligase Rsp5. Hse1 is linked to Rsp5 directly via a PY element within its C-terminus and through a novel protein Hua1, which recruits a complex of Rsp5, Rup1, and Ubp2. The SH3 domain of Hse1 also binds to the deubiquitinating protein Ubp7. Functional analysis shows that when both modes of Rsp5 association with Hse1 are altered, sorting of cargo that requires efficient ubiquitination for entry into the MVB is blocked, whereas sorting of cargo containing an in-frame addition of ubiquitin is normal. Further deletion of Ubp7 restores sorting of cargo when the Rsp5:Hse1 interaction is compromised suggesting that both ubiquitin ligases and peptidases associate with the Hse1-Vps27 sorting complex to control the ubiquitination status and sorting efficiency of cargo proteins. Additionally, we find that disruption of UBP2 and RUP1 inhibits MVB sorting of some cargos suggesting that Rsp5 requires association with Ubp2 to properly ubiquitinate cargo for efficient MVB sorting.     

Direct binding to Rsp5 mediates ubiquitin-independent sorting of Sna3 via the multivesicular body pathway

The sorting of most integral membrane proteins into the lumenal vesicles of multivesicular bodies (MVBs) is dependent on the attachment of ubiquitin (Ub) to their cytosolic domains. However, Ub is not required for sorting of Sna3, an MVB vesicle cargo protein in yeast. We show that Sna3 circumvents Ub-mediated recognition by interacting directly with Rsp5, an E3 Ub ligase that catalyzes monoubiquitination of MVB vesicle cargoes. The PPAY motif in the C-terminal cytosolic domain of Sna3 binds the WW domains in Rsp5, and Sna3 is polyubiquitinated as a consequence of this association. However, Ub does not appear to be required for transport of Sna3 via the MVB pathway because its sorting occurs under conditions in which its ubiquitination is impaired. Consistent with Ub-independent function of the MVB pathway, we show by electron microscopy that the formation of MVB vesicles does not require Rsp5 E3 ligase activity. However, cells expressing a catalytically disabled form of Rsp5 have a greater frequency of smaller MVB vesicles compared with the relatively broad distribution of vesicles seen in MVBs of wild-type cells, suggesting that the formation of MVB vesicles is influenced by Rsp5-mediated ubiquitination.     

Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase

Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery.     

Membrane-associated RING-CH 10 (MARCH10 protein) is a microtubule-associated E3 ubiquitin ligase of the spermatid flagella

Spermiogenesis is a complex and dynamic process of the metamorphosis of spermatids into spermatozoa. There is a great deal that is still unknown regarding the regulatory mechanisms for the formation of the sperm flagellum. In this study, we determined that the membrane-associated RING-CH 10 (March10) gene is predominantly expressed in rat testis. We isolated two March10 isoforms encoding MARCH10a and MARCH10b, which are generated by alternative splicing. MARCH10a is a long RING finger protein, and MARCH10b is a short RING finger-less protein. Immunohistochemical staining revealed that the MARCH10 proteins are specifically expressed in elongating and elongated spermatids, and the expression is absent in epididymal spermatozoa. MARCH10 immunoreactivity was observed in the cytoplasmic lobes as well as the principal piece and annulus of the flagella. When overexpressed in COS7 cells, MARCH10a was localized along the microtubules, whereas MARCH10b was distributed throughout the cytoplasm. An in vitro microtubule cosedimentation assay showed that MARCH10a is directly associated with microtubules. An in vitro ubiquitination assay demonstrated that the RING finger domain of MARCH10a exhibits an E3 ubiquitin ligase activity along with the E2 ubiquitin-conjugating enzyme UBE2B. Moreover, MARCH10a undergoes proteasomal degradation by autoubiquitination in transfected COS7 cells, but this activity was abolished upon microtubule disassembly. These results suggest that MARCH10 is involved in spermiogenesis by regulating the formation and maintenance of the flagella in developing spermatids.     

MARCH7 E3 ubiquitin ligase is highly expressed in developing spermatids of rats and its possible involvement in head and tail formation

Spermatogenesis is a highly complicated metamorphosis process of male germ cells. Recent studies have provided evidence that the ubiquitin–proteasome system plays an important role in sperm head shaping, but the underlying mechanism is less understood. In this study, we localized membrane-associated RING-CH (MARCH)7, an E3 ubiquitin ligase, in rat testis. Northern blot analysis showed that March7 mRNA is expressed ubiquitously but highly in the testis and ovary. In situ hybridization of rat testis demonstrated that March7 mRNA is expressed weakly in spermatogonia and its level is gradually increased as they develop. Immunohistochemical analysis detected MARCH7 protein expression in spermiogenic cells from late round spermatids to elongated spermatids and in epididymal spermatozoa. Moreover, MARCH7 was found to be localized to the caudal end of the developing acrosome of late round and elongating spermatids, colocalizing with β-actin, a component of the acroplaxome. In addition, MARCH7 was also detected in the developing flagella and its expression levels were prominent in elongated spermatids. We also showed that MARCH7 catalyzes lysine 48 (K48)-linked ubiquitination. Immunolocalization studies revealed that K48-linked ubiquitin chains were detected in the heads of elongating spermatids and in the acrosome/acroplaxome, neck, midpiece and cytoplasmic lobes of elongated spermatids. These results suggest that MARCH7 is involved in spermiogenesis by regulating the structural and functional integrity of the head and tail of developing spermatids.     

Ubiquitination and Degradation of CFTR by the E3 Ubiquitin Ligase MARCH2 through Its Association with Adaptor Proteins CAL and STX6

Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.     

A novel family of membrane-bound E3 ubiquitin ligases

A novel E3 ubiquitin ligase family that consists of viral E3 ubiquitin ligases (E3s) and their mammalian homologues was recently discovered. These novel E3s are membrane-bound molecules that share the secondary structure and catalytic domain for E3 activity. All family members have two transmembrane regions at the center and a RING-CH domain at the amino terminus. Forced expression of these novel E3s has been shown to reduce the surface expression of various membrane proteins through ubiquitination of target molecules. Initial examples of viral E3s were identified in Kaposi's sarcoma associated herpesvirus (KSHV) and murine gamma-herpesvirus 68 (MHV-68) and have been designated as modulator of immune recognition (MIR) 1, 2 and mK3, respectively. MIR 1, 2 and mK3 are able to down-regulate MHC class I molecule expression, and mK3 is required to establish an effective latent viral infection in vivo. The first characterized mammalian homologue to MIR 1, 2 and mK3 is c-MIR/MARCH VIII. Forced expression of c-MIR/MARCH VIII down-regulates B7-2, a co-stimulatory molecule important for antigen presentation. Subsequently, several mammalian molecules related to c-MIR/MARCH VIII have been characterized and named as membrane associated RING-CH (MARCH) family. However, the precise physiological function of MARCH family members remains as yet unknown.     

gamma2-Adaptin, a ubiquitin-interacting adaptor, is a substrate to coupled ubiquitination by the ubiquitin ligase Nedd4 and functions in the endosomal pathway

gamma2-Adaptin is a putative member of the clathrin adaptor protein family with unknown physiological function. We previously reported that gamma2-adaptin acts as a ubiquitin receptor by virtue of its ubiquitin-interacting motif. Here we demonstrate that this motif mediates a specific physical interaction with the ubiquitin ligase Nedd4 and promotes ubiquitination of gamma2-adaptin. By mapping regions of Nedd4 involved in binding to gamma2-adaptin, we identified its C2 domain to be essential, whereas the WW and HECT domains are dispensable. Consistent with this, we uncovered that the C2 domain of Nedd4 is ubiquitinated itself and as such is recruited by the ubiquitin-interacting motif of gamma2-adaptin for subsequent ubiquitin conjugation. Unlike known coupled ubiquitination reactions, this novel type of interaction leads to mono- and multi/polyubiquitinated gamma2-adaptin. In addition, we show that gamma2-adaptin functions in the endosomal/multivesicular body (MVB) pathway. Depletion of gamma2-adaptin impairs the degradation of internalized epidermal growth factor and results in defective MVB morphology characterized by significantly enlarged vesicles. These defects cannot be rescued by gamma1-adaptin, a closely related homolog of gamma2-adaptin, which is unable to bind ubiquitin. Together, these results indicate that gamma2-adaptin may operate within the MVB sorting system in a manner different from that of classic adaptins.     

Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1

The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1.     

TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.     

MARCH6 promotes Papillary Thyroid Cancer development by destabilizing DHX9

Membrane-associated ring-CH-type finger (MARCH) proteins belong to the E3 ubiquitin ligase family, which regulates protein stability by increasing ubiquitination. Recent evidence has shown that some MARCH proteins play important roles in cancer development. However, the role of MARCH6 in tumorigenesis, including thyroid tumorigenesis, remains unknown. In this study, we determined that MARCH6 was upregulated in the majority of primary papillary thyroid cancers (PTCs) at both the mRNA and protein levels. Gain-of-function and loss-of-function studies demonstrated that MARCH6 suppressed apoptosis and promoted cell cycle progression, cell proliferation, growth, migration and tumorigenesis in thyroid cancer cells. Mechanistically, MARCH6 interacted with and downregulated DHX9. Knockdown of DHX9 enhanced the proliferative and migratory abilities of thyroid cancer cells. The inhibitory effect of MARCH6 knockdown on thyroid cancer cell growth and migration was also reversed by DHX9 silencing. In addition, MARCH6 activated the AKT/mTOR signaling pathway in a manner dependent on the downregulation of DHX9. Overall, MARCH6 functions as a potential oncogene in thyroid cancer by destabilizing DHX9 and activating AKT/mTOR signaling.     

Direct binding to Rsp5p regulates ubiquitination-independent vacuolar transport of Sna3p

The sorting of integral membrane proteins such as carboxypeptidase S (Cps1p) into the luminal vesicles of multivesicular bodies (MVBs) in Saccharomyces cerevisiae requires ubiquitination of their cytosolic domains by the ubiquitin ligases Rsp5p and/or Tul1p. An exception is Sna3p, which does not require ubiquitination for entry into MVBs. The mechanism underlying this ubiquitination-independent MVB sorting pathway has not yet been characterized. Here, we show that Sna3p sorting into the MVB pathway depends on a direct interaction between a PPAY motif within its C-terminal cytosolic tail and the WW domains of Rsp5p. Disruption of this interaction inhibits vacuolar targeting of Sna3p and causes its accumulation in a compartment that overlaps only partially with MVBs. Surprisingly, Sna3p does require a functional ubiquitin-ligase HECT domain within Rsp5p; however, the dependence of Sna3p on HECT domain activity is distinct from that of Cps1p. Last, we show that Sna3p requires neither Tul1p nor the transmembrane adaptor protein Bsd2p for its MVB sorting. Our data demonstrate that Sna3p follows a novel ubiquitination-independent, but Rsp5p-mediated, sorting pathway to the vacuole.     

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.     

The PC6B cytoplasmic domain contains two acidic clusters that direct sorting to distinct trans-Golgi network/endosomal compartments

The mammalian proprotein convertases (PCs) are a family of secretory pathway enzymes that catalyze the endoproteolytic maturation of peptide hormones and many bioactive proteins. Two PCs, furin and PC6B, are broadly expressed and share very similar cleavage site specificities, suggesting that they may be functionally redundant. However, germline knockout studies show that they are not. Here we report the distinct subcellular localization of PC6B and identify the sorting information within its cytoplasmic domain (cd). We show that in neuroendocrine cells, PC6B is localized to a paranuclear, brefeldin A-dispersible, BaCl(2)-responsive post-Golgi network (TGN) compartment distinct from furin and TGN38. The 88-amino acid PC6B-cd contains sorting information sufficient to direct reporter proteins to the same compartment as full-length PC6B. Mutational analysis indicates that endocytosis is predominantly directed by a canonical tyrosine-based motif (Tyr(1802)GluLysLeu). Truncation and sufficiency studies reveal that two clusters of acidic amino acids (ACs) within the PC6B-cd contain differential sorting information. The membrane-proximal AC (AC1) directs TGN localization and interacts with the TGN sorting protein PACS-1. The membrane-distal AC (AC2) promotes a localization characteristic of the full-length PC6B-cd. Our results demonstrate that AC motifs can target proteins to distinct TGN/endosomal compartments and indicate that the AC-mediated localization of PC6B and furin contribute to their distinct roles in vivo.