Molecular characterization of mammalian homologues of Class C Vps proteins that interact with syntaxin-7

localizations


Summary
Vesicle-mediated protein sorting plays an important role in segregation of intracellular molecules into distinct organelles. Extensive genetic studies using yeast have identified more than forty VPS genes involved in vesicle transport to vacuoles. However, their mammalian counterparts are not fully elucidated. In this study, we identified two human homologues of yeast Class C VPS genes, human VPS11 (hVPS11) and human VPS18 (hVPS18). We also characterized the subcellular localization and interactions of the protein products not only from these genes but also from the other mammalian Class C VPS homologue genes, hVPS16, and rVPS33a. The protein products of hVPS11 (hVps11) and hVPS18 (hVps18) were ubiquitously expressed in peripheral tissues, suggesting that they have a fundamental role in cellular function. Indirect immunofluorescence microscopy revealed that the mammalian Class C Vps proteins are predominantly associated with late endosomes/lysosomes. Immunoprecipitation and gel filtration studies showed that the mammalian Class C Vps proteins constitute a large hetero-oligomeric complex that interacts with syntaxin-7. These results indicate that, like their yeast counterparts, mammalian Class C Vps proteins mediate vesicle trafficking steps in the endosome/lysosome pathway.

Introduction
Eukaryotic cells contain highly specialized intracellular membrane bound compartments. Vesicle trafficking between these organelles is very important for the maintenance of cell homeostasis (1)(2). Although numerous proteins and protein complexes have been characterized to have a role in intracellular vesicle transport and protein sorting, their precise mechanisms of involvement are not yet elucidated. Genetic studies using yeast mutants have identified more than 40 vacuolar protein sorting (VPS) genes coding for proteins required for vacuolar proteins transports (3)(4)(5). These vps mutants are categorized into six classes, A-F, with respect to their morphology and acidification defects (6,7). The Class C vps mutants are characterized by remarkable abnormalities in vacuole morphology, accumulations of multivesicular bodies, temperature sensitive growth defects, osmotic sensitivity, reduced amino acid pools and sporulation defects (5,6,(8)(9)(10)(11).
Previous studies have reported that Vps18p and Vps33p share significant homology with the Drosophila gene products, deep orange (dor) and carnation (car), respectively. These proteins are associated into a large complex that localizes to the endosomal compartment and is required for membrane trafficking to lysosomes and pigment granules in Drosophila eyes (20,21). Therefore it appeared likely that mammalian homologues of the yeast Class C Vps proteins are also involved in protein sorting steps. Additionally, syntaxin-7, a Vam3p related protein was recently identified in mammals (22)(23)(24)(25). Although there is some discrepancy regarding the precise intracellular localization of syntaxin-7, it is clear that syntaxin-7 is an essential factor for the fusion of late endosomes with lysosomes, lysosome homotypic fusion, and endocytic trafficking to late endosomes (24)(25)(26).
The functional roles of Class C Vps proteins have been extensively investigated in yeast. In contrast, relatively little is known about their mammalian counterparts. Therefore, we sought to identify and characterize Vps proteins that may control intracellular vesicle trafficking events in mammalian cells. In the present study, we identify two human VPS gene homologues, hVPS11 and hVPS18, and characterize biochemical features and intracellular localizations of Class C Vps proteins. We show that mammalian Class C Vps proteins exist as a hetero-oligomeric complex primarily associated with late endosomes/lysosomes, and that the complex interacts with syntaxin-7 suggesting that it has a role in SNARE complex assembly.

Experimental procedures
Isolation of hVPS11 and hVPS18----The GenBank TM database of human expressed sequence tags (ESTs) was searched using the S. cerevisiae VPS11/PEP5/END1 sequences (8,10). One of the human EST clones (accession number: AA385518) had a distant homology to the RING-H2 finger domain region of S. cerevisiae VPS11/PEP5/END1. Two oligonucleotide PCR primers (5'-AGCAGATTGCACAGGATGAG-3' and 5'-CAGAGTCAATTTGTTGAAAA-3') were designed and used to amplify a 395 bps fragment using a HeLa cell cDNA template for polymerase chain reaction (PCR) under standard conditions. Amplified fragments were subcloned into pGEM-T Easy vector (Promega, Madison, WI) and subsequently sequenced. The EcoRI digested insert fragment from pGEM-T Easy vector was used as a probe for screening the human brain cDNA library constructed in lambda ZAPII (gift from Dr. S. Nakanishi, Kyoto University, Kyoto, Japan). Ten positive clones were obtained from 1 X 10 6 plaques screened. The clone carrying the longest insert was sequenced from both strands.
hVPS18 was identified by searching the GenBank TM database using S. cerevisiae VPS18/PEP3 sequences (11,13) and Drosophila deep orange sequences (20,21). A novel cDNA, designated KIAA1475 (accession number: AB040908) containing an open reading frame of 976 amino acids was found to be 34% identical to deep orange and 26% identical to S. cerevisiae VPS18/PEP3. The KIAA1475 clone was obtained from the KAZUSA DNA institute (Chiba, Japan).
Northern Blot Analyses----A hVPS11 cDNA fragment was excised using the BamHI restriction enzyme (1,808 bps), and a hVPS18 cDNA fragment was excised using the SacI restriction enzyme (972 bps). Fragments were randomprimed radiolabeled (Life Technologies, Rockville, MD) and hybridization was carried out at 42°C overnight using human multiple tissue northern (MTN) blots (Clontech, Palo Alto, CA) as described previously (27).
Expression Vectors----Epitope-tagged, full-length hVPS11 and hVPS18 and rVPS33a were prepared using PCR with custom designed oligonucleotide primers containing the appropriate restriction enzyme sites. To insert full length hVPS11 downstream of the Myc-and HA-tag sequence at the N-terminal end, a forward primer containing the EcoRI site upstream of the initiation codon (5'-CGGAATTCAAATGGCGGCCTACCTGCA-3') and a reverse primer including the XhoI site downstream of stop codon (5'-CCTCGAGTTAAGTGCCCCTCCTGGA-3') were used to amplify PCR products from a pBluescript SK(+)-hVPS11 template.
PCR products were subcloned into the pGEM-T Easy vector and subsequently sequenced. The EcoRI/XhoI digested full-length hVPS11 was inserted into the EcoRI and XhoI site of the pMyc-CMV and pHA-CMV mammalian expression vectors (Clontech) to generate pMyc-and pHA-hVPS11, respectively. The Nterminally Myc-and HA-tagged hVPS18 constructs were also generated by PCR using oligonucleotide primers, 5' primer-CGGAATTCCCATGGCGTCCATCCAT, 3' primer-CCGCTCGAGCTACAGCCAACTGAGC using pBluescript II SK(+)-KIAA1475 clone as a template. EcoRI and XhoI digested full-length hVPS18 was inserted into the EcoRI and XhoI site of the pMyc-CMV and pHA-CMV mammalian expression vectors to generate pMyc-and pHA-hVPS18, respectively. A full-length rat VPS33a (accession number: U35244) was amplified by PCR from rat total brain cDNAs. A forward primer containing the XhoI site upstream of the initiation codon (5'-CAGATCTCGAGCGATGGCGGCTCACCT) and a reverse primer containing the EcoRI site downstream of the stop codon (5'-CAGAATTCCTAGAAAGGCTTTTCCATGA) were used to amplify PCR products.
The XhoI and EcoRI digested full-length rVPS33a was inserted into the XhoI and EcoRI site of the pEGFP-C1 mammalian expression vector (Clontech) to generate GFP-rVPS33a. The N-terminally Myc-tagged full-length human syntaxin-7 (accession number: U77942) construct was generated by PCR using custom designed oligonucleotide primers. The 5'-primer (CGGAATTCCCATGTCTTACACTCCA) and the 3'-primer (AATGCGGCCGCTCAGTGGTTCAATC) were used to amplify PCR products from a human cDNA brain library. All constructs were verified by DNA sequencing.
The blots were incubated with anti-hVps11, hVps16, and hVps18 antibodies for 2 hr at room temperature and then incubated in HRP-conjugated secondary antibodies. Antibody binding was detected using the ECL protein detection kit (Amersham Pharmacia Biotech) according to the manufacturer specifications.

Results
Cloning of human homologues of Yeast VPS11 and VPS18----To identify mammalian homologues of the yeast Class C VPS genes, we searched the expressed sequence tag (EST) database using the BLAST (National Center for Biotechnology Information, Bethesda, MD) with the S. cerevisiae Vps11p as a query. A human EST clone (accession number: AA385518) fragment encoding a RING-H2 finger domain was identified. This cDNA fragment was used to probe a human brain cDNA library. Among the ten positive clones, the longest ORF was 2,823 bp encoding a 941-amino acid polypeptide (Fig. 1A). A BLAST search for the NR protein database using this ORF amino acid sequence as the query retrieved not only S. cerevisiae Vps11p but also homologues of other species at the high score range. We hereafter refer to this protein as human Vps11 (hVps11).
The human protein homologue of yeast Vps18p, hVps18, was identified by searching the sequence database using S. cerevisiae Vps18p and the Drosophila dor protein as a query. The KIAA1475 cDNA (accession number: AB040908), was found contain an open reading frame of 976-amino acids (Fig. 1B), which displays 34% overall identity Drosophila dor protein and 26% identity with S. cerevisiae from all the cell lines tested (Fig. 3A, B). The anti-hVps18 antibody recognized an additional higher molecular weight band in COS-7 and BHK cells and an approximately 88 kDa protein band in HeLa, HEK293, COS-7 and NRK cell lines.
This 88 kDa band may represent a hVps18 degradation product since it was not detected in membrane fractionation experiments (see Fig. 4A). The anti-hVps16 antibody recognized a major protein band migrating at approximately 97kDa in lysates from all cell lines tested (Fig. 3C). These results suggest that the human Class C Vps proteins cycle between the cytosolic and membrane-bound pools. hVps11, hVps16 and hVps18 are membrane-associated----Although hVps11, hVps16 and hVps18 lack a putative transmembrane region, they are found to be in the membrane fractions from HEK293 cells. To elucidate the molecular mechanism of their membrane association, membrane fractions from HEK293 cells were further extracted with 1.5 M NaCl, 5 M urea, 0.2 M sodium bicarbonate at pH 11.4, or 1% Triton X-100, respectively. The extracts were Western blotted for hVps11, hVps16, and hVps18. All three proteins were highly solubilized with urea, sodium bicarbonate and Triton X-100, but only partially extracted with in 1.5 M NaCl (Fig. 4B). These results suggest that the proteins are primarily soluble and associated weakly with the cytosolic face of membranes. hVps11, hVps16, and hVps18 associate with late endosomes/lysosomes----To further determine the intracellular localizations of hVps11, hVps16, and hVps18, immunocytochemistry was performed on COS-7 cells. As shown in Fig.   5, all three proteins showed a vesicular and cytosolic staining. Staining was eliminated when antibodies were preincubated with the respective antigen (data not shown). Essentially the same stainings were observed in HeLa, NRK and BHK cells (data not shown). We then compared the immunostaining patterns for hVps11, hVps16, and hVps18 with those of EEA1, transferrin receptor (TfR), and lysosome associated membrane protein-1 (Lamp-1). EEA1, TfR and Lamp-1 were selected for comparison as standard markers for early endosomes (29,30), recycling endosomes/plasma membranes (31,32), and late endosomes/lysosomes (33), respectively. The staining patterns of hVps11, hVps16, and hVps18 overlapped significantly with that of Lamp-1 (Fig. 5). These observations indicate that human Class C Vps proteins are associated primarily with late endosomes/lysosmes, and are compatible with the fact that their yeast counterparts are involved in the late step transport to vacuoles.

Human Class C Vps proteins interact with each other in vivo----
Previous studies have shown that yeast Class C Vps proteins form a complex within cells (12,(16)(17)(18). To test whether such a complex is formed also in mammalian cells, interactions of endogenous human Class C Vps proteins were examined by immunoprecipitation experiments. As shown in Fig. 6A, anti-hVps11 antibody co-immunoprecipitated hVps18 and hVp16. Similarly, antibodies against hVps18 and hVps16 co-immunoprecipitated the other two proteins ( Fig. 6B and C, respectively). These data unequivocally indicate that those three proteins interact with each other in vivo. hVps11, hVps18, and rVps33a are constitute a large oligomeric complex----We also analyzed whether the other mammalian Class C Vps protein, rVps33a is included in the complex. For this purpose, expression vectors for Myc-, HAand GFP tagged mammalian Class C Vps proteins were cotransfected into COS-7 cells. HA-hVps18 and GFP-rVps33a efficiently co-immunoprecipitated with Myc-hVps11 (Fig. 7A). Similarly, Myc-hVps11 and hVps18 co-immunoprecipitated with GFP-rVps33a (Fig. 7B).
GFP-rVps33a demonstrated a vesicular staining pattern and colocalized with hVps11 immunoreactivity (Fig. 7C). Similar colocalization was observed for hVps18 and hVps16 (data not shown).
In order to analyze whether mammalian Class C Vps proteins constitute a complex in vivo, COS-7 cells expressing myc-hVps11p, HA-hVPS18p, and GFP-rVps33a were subjected to gel filtration analyses. In cytosolic fraction of transfected COS-7 cells, these three molecules migrated to the fractions corresponding to high molecular masses (>670 kDa), suggesting that they form a large oligomeric protein complex (Fig. 7D).
Taken together with the data in Fig. 6 and Fig. 7, these results indicate that mammalian Class C Vps proteins constitute a hetero-oligomeric complex in vivo, and play roles for late endosome/lysosomal trafficking pathway. hVps11 and hVps18 are associated with syntaxin-7----Previous reports have described that the Class C Vps complex binds to Vam3p but not Vam7p or Vti1 suggesting that it may function prior to trans-SNARE pairing and is required for vesicle docking/fusion reactions (16,19). It has also been reported that the mammalian counterpart of yeast Vam3p, syntaxin-7, is responsible for mediating endocytic trafficking to late endosomes as well as late endosome-lysosome and lysosome hetero/homotypic fusion (24)(25)(26). To examine whether this interaction is conserved between yeast and mammals, Myc-tagged human syntaxin-7 (Myc-hSyn-7) and either HA-tagged hVPS11or hVPS18 were transfected into COS-7 cells and subjected to immunoprecipitation analyses. Myc-hSyn-7 was coimmunoprecipitated with both HA-hVps11 and HA-hVps18 (Fig. 8A). Similarly, when the inverse immunoprecipitation was performed, HA-tagged hVps11 and hVps18 were found to co-immunoprecipitate with Myc-hSyn-7 (Fig. 8B).
To verify this result, Myc-hSyn-7 transfected COS-7 cells were immunostained anti-hVps11, hVps18, and hVps16 antibody. Myc-hSyn-7 showed a vesicular staining pattern and colocalized with hVps11 immunoreactivity in some vesicle structures (Fig. 8C). Similar localization was observed for hVps18 and hVps16 (data not shown). This interaction of mammalian Class C Vps proteins and syntaxin-7 indicate that mammalian Class C Vps complex functions are at the late endocytic trafficking and membrane docking/fusion reactions of late endosomes/lysosomes.

Discussion
The yeast Class C Vps proteins form a large hetero-oligomeric protein complex and mediate the delivery of vacuolar hydrolases to vacuoles and regulates homotypic vacuole fusion through interactions with Vam3p (12,(16)(17)(18)(19).
In the present study, we identified two human homologues of yeast Class C Vps proteins, hVps11 and hVps18. Furthermore, we showed that these proteins along with other Class C Vps proteins, hVps16 and rVps33a, constitute a heterooligomeric complex, interact with the Vam3p homologue, syntaxin-7, and associate en bloc with membranes of late endosomes/lysosomes.  (Fig. 1C). These domains are found in various proteins that form multiprotein complexes, including those with known roles in membrane trafficking (14,15). EEA1 has a specific lipid-binding domain, FYVE finger, which is also a variant of the RING finger domain. The FYVE finger domain binds to PtdIns(3)P on early endosome membranes with high specificity and is required for early endosome localization of EEA1 (34)(35)(36)(37). It is likely that the RING-H2 finger domains of hVps11 and hVps18 are also involved in protein-protein and/or protein-lipid interactions. In addition, hVps11 and hVps18 are predicted to form one or more α-helical coiled-coil domain (38). The coiled-coil domain is conserved in a broad spectrum of membrane fusion proteins, suggesting a similar function for this domain in the Class C Vps proteins (39). Finally, hVps11 and hVps18 have a highly conserved sequence related to a region of the clathrin heavy chain repeat (CHCR) domains required for clathrin heavy chain self assembly, and light chain binding and trimerization (Fig. 1A, B). The CHCR domains are also found in other proteins implicated in vacuole protein sorting, such as Vam2p/Vps41p and Vam6p/Vps39p (40,41), suggesting that the domains are responsible for their complex formation. We are currently investigating the roles of these domains in the complex formation and association with their target membranes of the mammalian Class C Vps proteins.
Northern blot and immunoblot analyses of the mammalian Vps proteins showed that they are expressed in a wide variety of tissues and cell lines, suggesting that these proteins may have common physiological functions.
Previous studies in yeast demonstrated that Class C Vps proteins cofractionated with vacuolar membranes (12). Our cellular fractionation and subcellular localization studies showed that mammalian Class C Vps proteins are soluble proteins that are weakly associated with the cytosolic face of endosome/lysosome membranes. Furthermore, our immunoprecipitation and gel filtration analyses unequivocally demonstrated that all the mammalian Class C Vps proteins together constitute a hetero-oligomeric protein complex like the yeast counterparts.
Homologues of Vps18p and Vps33p have been also identified in Drosophila; Dor and Car, respectively, whose mutations cause defects in eye pigmentation (20,21). Car and Vps33p belong to the family of Sec1p-related proteins, which are essential for vesicle docking and fusion by binding to the syntaxin like SNAREs (21,42). Biochemical studies have demonstrated that Dor and Car are part of a large protein complex associated with endosomal membranes. Phenotypic characterization of the dor and car mutants has indicated defects in the lysosomal delivery of internalized ligands and the biogenesis of the pigment granules, a compartment related to the vacuole/lysosomes (21). Collectively, the organization and functions of the Class C Vps protein complex appear to be conserved from yeast through Drosophila to mammals.
Several lines of evidence have suggested that, in yeast, the molecular complex including Class C Vps proteins contain two additional regulators of vacuolar fusion and docking, Vam2p/Vps41p and Vam6p/Vps39p. This complex was termed HOPS (homotypic fusion and vacuole protein sorting) complex and is essential for homotypic vacuole fusion and vacuole protein sorting (17,18).
Although it is not entirely clear whether mammalian Class C Vps proteins interact with the counterparts of Vam2p/Vps41p and Vam6p/Vps39p, the structural similarities between the yeast and mammalian Class C Vps proteins suggest that this may be the case. Syntaxin-7 shares 24% identity with yeast Vam3p and is ubiquitously expressed in multiple tissues tested (22). It is localized to the late endosomes and is required for late endosome and lysosome fusion. Induced expression of mouse syntaxin-7 lacking the transmembrane domain block endocytic transport from early endosome to late endosome (24,25). Likewise, microinjection into cells of bacterially expressed syntaxin-7 lacking the transmembrane domain or of antisyntaxin-7 antibodies inhibits homotypic lysosome fusion and heterotypic late endosome/lysosome fusion but has no affect on homotypic early endosome fusion (26). Although there is some discrepancy regarding the localization of mammalian syntaxin-7, the syntaxin-7 expression pattern is very similar to that of hVps11 and hVps18, and both hVps11 and hVps18 interact with Myc-tagged human syntaxin-7. Taken together, it seems likely that mammalian Class C Vps proteins are also required for late endosome/lysosome fusion and the endocytic transport pathway.
Many questions still remain regarding the roles of mammalian Class C Vps proteins. Our results suggest that Class C Vps proteins are structurally and functionally conserved among yeast, Drosophila and mammals. Understanding the interaction of mammalian Class C Vps proteins with syntaxin-7 will likely provide significant clues for studying membrane docking and fusion events in mammalian cells. Figures   Fig. 1. Amino acid sequences and