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A Human Homolog Can Functionally Replace the Yeast Vesicle-associated SNARE Vti1p in Two Vesicle Transport Pathways

1998; Elsevier BV; Volume: 273; Issue: 5 Linguagem: Inglês

10.1074/jbc.273.5.2624

1083-351X

Gabriele Fischer von Mollard, Tom H. Stevens,

Endoplasmic Reticulum Stress and Disease

Membrane traffic in eukaryotic cells requires the interaction of a vesicle-associated soluble NSF attachment protein receptor (v-SNARE) on transport vesicles with a SNARE on the target membrane (t-SNARE). Recently, we identified the yeast protein Vti1p as a v-SNARE that is involved in two transport reactions. Vti1p interacts with the prevacuolar t-SNARE Pep12p in Golgi to prevacuolar transport and with the cis-Golgi t-SNARE Sed5p in traffic to the cis-Golgi. Here we describe a human Vti1p homolog, hVti1. Whereas vti1Δcells are inviable, expression of hVti1 allows vti1Δcells to grow at nearly the wild-type growth rate. When expressed in yeast hVti1 can replace Vti1p in both Golgi to prevacuolar transport and in traffic to the cis-Golgi. Sequence comparisons with aSchizosaccharomyces pombe and two different mouse Vti1 homologs led to the identification of a very conserved predicted α-helix. Amino acid exchanges in vti1 mutant alleles defective either in one or both trafficking steps cluster in this domain, suggesting that this structure is probably the binding site for effector proteins. Membrane traffic in eukaryotic cells requires the interaction of a vesicle-associated soluble NSF attachment protein receptor (v-SNARE) on transport vesicles with a SNARE on the target membrane (t-SNARE). Recently, we identified the yeast protein Vti1p as a v-SNARE that is involved in two transport reactions. Vti1p interacts with the prevacuolar t-SNARE Pep12p in Golgi to prevacuolar transport and with the cis-Golgi t-SNARE Sed5p in traffic to the cis-Golgi. Here we describe a human Vti1p homolog, hVti1. Whereas vti1Δcells are inviable, expression of hVti1 allows vti1Δcells to grow at nearly the wild-type growth rate. When expressed in yeast hVti1 can replace Vti1p in both Golgi to prevacuolar transport and in traffic to the cis-Golgi. Sequence comparisons with aSchizosaccharomyces pombe and two different mouse Vti1 homologs led to the identification of a very conserved predicted α-helix. Amino acid exchanges in vti1 mutant alleles defective either in one or both trafficking steps cluster in this domain, suggesting that this structure is probably the binding site for effector proteins. Transport between many different organelles in eukaryotic cells occurs via transport vesicles, which must have the ability to recognize their target membranes. SNARE 1The abbreviations used are: SNARE, soluble NSF attachment protein receptor; v-SNARE, vesicle-associated SNARE; t-SNARE, target membrane-associated SNARE; CPY, carboxypeptidase Y; ER, endoplasmic reticulum; EST, expressed sequence tag; PCR, polymerase chain reaction; kb, kilobase pair(s). proteins provide this information (1Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2011) Google Scholar, 2Ferro-Novick S. Jahn R. Nature. 1994; 370: 191-193Crossref PubMed Scopus (561) Google Scholar). In the SNARE model, a specific set of v-SNAREs localized on transport vesicles interacts with specific t-SNAREs on the target membrane. Both v- and t-SNAREs contain a single C-terminal transmembrane domain and predicted coiled coil domains. It has been demonstrated that t-SNAREs interact via their coiled coil domains with v-SNAREs. A growing number of SNARE proteins have been identified from both yeast and mammalian cells. SNARE proteins involved in identical membrane trafficking steps in yeast and mammals share significant amino acid identities. In yeast the v-SNAREs Sec22p (Sly2p), Bet1p (Sly12p), Bos1p, and Ykt6p are involved in transport from the ER to the cis-Golgi compartment (3Newman A.P. Shim J. Ferro-Novick S. Mol. Cell. Biol. 1990; 10: 3405-3414Crossref PubMed Scopus (141) Google Scholar, 4Ossig R. Dascher C. Trepte H.H. Schmitt H.D. Gallwitz D. Mol. Cell. Biol. 1991; 11: 2980-2993Crossref PubMed Scopus (149) Google Scholar, 5McNew J.A. Søgaard M. Lampen N.M. Machida S. Ye R.R. Lacomis L. Tempst P. Rothman J.E. Söllner T.H. J. Biol. Chem. 1997; 272: 17776-17783Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Their t-SNARE partner in the cis-Golgi compartment is Sed5p (6Hardwick K.G. Pelham H.R. J. Cell Biol. 1992; 119: 513-521Crossref PubMed Scopus (256) Google Scholar). It has been demonstrated that in addition to interactions with the anterograde v-SNAREs, Sed5p also binds to the medial Golgi v-SNARE Sft1p, which is involved in retrograde traffic from the medial to the cis Golgi compartment (7Banfield D.K. Lewis M.J. Pelham H.R. Nature. 1995; 375: 806-809Crossref PubMed Scopus (129) Google Scholar). Recently, Sec22p has been found in a complex with the ER t-SNARE Ufe1p and has been implicated in retrograde traffic to the ER (8Lewis M.J. Rayner J.C. Pelham H.R.B. EMBO J. 1997; 16: 3017-3024Crossref PubMed Scopus (122) Google Scholar). Mammalian homologs have been identified for Sec22p, rsec22 and ERS-24, and for Bet1p, rbet1 (9Hay J.C. Hirling H. Scheller R.H. J. Biol. Chem. 1996; 271: 5671-5679Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 10Paek I. Orci L. Ravazolla M. Erdjument-Bromage H. Amherdt M. Tempst P. Söllner T.H. Rothman J.E. J. Cell Biol. 1997; 137: 1017-1028Crossref PubMed Scopus (43) Google Scholar). ERS-24 interacts with syntaxin 5, the mammalian homolog of Sed5p (11Dascher C. Matteson J. Balch W.E. J. Biol. Chem. 1994; 269: 29363-29366Abstract Full Text PDF PubMed Google Scholar). Proteins traversing the secretory pathway are sorted in the trans-Golgi network according to their destination (12Pryer N.K. Wuestehube L.J. Schekman R. Annu. Rev. Biochem. 1992; 61: 471-516Crossref PubMed Scopus (369) Google Scholar). In mammalian cells, vesicles of the constitutive and regulated secretory pathway destined for the plasma membrane bud from the trans-Golgi network. Soluble lysosomal proteins are marked by a mannose 6-phosphate residue and bind to the mannose 6-phosphate receptor (13Kornfeld S. Annu. Rev. Biochem. 1992; 61: 307-330Crossref PubMed Scopus (936) Google Scholar). The complex leaves in transport vesicles targeted to the late endosomal compartment, from which the mannose 6-phosphate receptor is recycled. In yeast about 50VPS and PEP genes have been identified, which function in traffic from the late Golgi compartment to the vacuole, the mammalian equivalents of the trans-Golgi network and the lysosome respectively (14Jones E.W. Genetics. 1977; 85: 23-33Crossref PubMed Google Scholar, 15Bankaitis V.A. Johnson L.M. Emr S.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9075-9079Crossref PubMed Scopus (280) Google Scholar, 16Rothman J.H. Stevens T.H. Cell. 1986; 47: 1041-1051Abstract Full Text PDF PubMed Scopus (299) Google Scholar, 17Stack J.H. Horazdovsky B.F. Emr S.D. Annu. Rev. Cell Dev. Biol. 1995; 11: 1-33Crossref PubMed Scopus (172) Google Scholar). The soluble vacuolar hydrolase carboxypeptidase Y (CPY) binds to the CPY-receptor Vps10p in the late Golgi compartment via a peptide sorting signal (18Marcusson E.G. Horazdovsky B.F. Cereghino J.L. Gharakhanian E. Emr S.D. Cell. 1994; 77: 579-586Abstract Full Text PDF PubMed Scopus (402) Google Scholar). The complex is transported to the prevacuolar compartment where it dissociates. CPY is transported on to the vacuole and Vps10p is recycled (19Piper R.C. Cooper A.A. Hong Y. Stevens T.H. J. Cell Biol. 1995; 131: 603-617Crossref PubMed Scopus (342) Google Scholar, 20Cereghino J.L. Marcusson E.G. Emr S.D. Mol. Biol. Cell. 1995; 6: 1089-1102Crossref PubMed Scopus (135) Google Scholar, 21Cooper A.A. Stevens T.H. J. Cell Biol. 1996; 133: 529-541Crossref PubMed Scopus (240) Google Scholar). Pep12p has been identified as a t-SNARE residing in the prevacuolar compartment (22Becherer K.A. Rieder S.E. Emr S.D. Jones E.W. Mol. Biol. Cell. 1996; 7: 579-594Crossref PubMed Scopus (253) Google Scholar). The mammalian protein syntaxin 6 displays 25% amino acid identity with Pep12p, is localized in the Golgi region, and has been proposed to be the functional homolog of Pep12p (23Bock J.B. Klumperman J. Davanger S. Scheller R.H. Mol. Biol. Cell. 1997; 8: 1261-1271Crossref PubMed Scopus (248) Google Scholar). A Pep12p homolog with 32% amino acid identity has been identified in Arabidopsis, which can restore at least some vacuolar protease function to pep12mutants in yeast (24Bassham D.C. Gal S. da Silva Conceicao A. Raikhel N.V. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7262-7266Crossref PubMed Scopus (90) Google Scholar). Recently, we described a yeast v-SNARE, Vti1p, that is essential for yeast cell viability (25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar). The temperature-sensitive vti1mutants vti1-1 and vti1-2 are defective in late Golgi to prevacuolar transport of CPY. Genetic interactions betweenVTI1 and PEP12 as well as physical association of Vti1p and Pep12p indicate that Vti1p and Pep12p form a v-SNARE-t-SNARE complex. A second class of temperature-sensitive mutants, such asvti1-11, display a severe growth defect and a block in traffic to the cis-Golgi compartment, in addition to a block in late Golgi to prevacuolar transport of CPY. Overexpression of Sed5p suppressed the cis-Golgi traffic block but had no effect on the defect in sorting proteins to the vacuole. Recombinant Vti1p and Sed5p interacted in vitro. We have proposed that Vti1p forms a SNARE complex with Sed5p in retrograde traffic either from the prevacuolar compartment or from the late Golgi compartment. Here we describe the identification of a human Vti1 homolog. hVti1 is able to function in both traffic steps that require Vti1p in yeast. Conserved amino acid residues between the yeast and human proteins together with mutations in yeast VTI1 have led us to propose a structural model, in which a predicted α-helical coiled coil domain in Vti1p interacts with both Pep12p and Sed5p. Reagents were used from the following sources: enzymes for DNA manipulation from New England Biolabs (Beverly, MA) and Boehringer Mannheim, [35S]Express label from NEN Life Science Products, fixed Staphylococcus aureus cells (IgGsorb) from The Enzyme Center (Malden, MA), and Oxalyticase from Enzogenetics (Corvallis, OR). The human hypothalamus cDNA library in λZAPII was obtained from the ATCC (Rockville, MD). All other reagents were purchased from Sigma. Yeast strains (Table I) were grown in rich medium (1% yeast extract, 1% peptone, 2% dextrose, YEPD) or standard minimal medium (SD) with appropriate supplements. To induce expression from the GAL1 promoter, dextrose was replaced by 2% raffinose and 2% galactose. Plasmid manipulations were performed in the Escherichia coli strains MC1061 or XL1Blue using standard media (Table II).Table IYeast strains used in this studyStrainGenotypeReferenceFvMY6MATα leu2–3,112 ura3–52 ade2–101 trp1-Δ901 suc2-Δ9 mel- vti1Δ::HIS3Fischer von Mollard et al.(25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar)FvMY7MAT a leu2–3,112 ura3–52 his3-Δ200 ade2–101 trp1-Δ901 suc2-Δ9 mel- vti1–1Fischer von Mollard et al. (25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar)FvMY21MAT a leu2–3,112 ura3–52 his3-Δ200 ade2–101 trp1-Δ901 suc2-Δ9 mel- vti1–11This study Open table in a new tab Table IIPlasmids used in this studyPlasmidDescriptionReferencepFvM16GAL1-VTI1::HAin pRS314 (CEN6-TRP1)Fischer von Mollard et al. (25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar)pFvM281.8 kb containing VTI1 in pRS314 (CEN6-TRP1)Fischer von Mollard et al. (25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar)pFvM46PCR-amplified hVti1 from pADANS in pBluescript KS+This studypFvM50pADANS vti1Δ suppressor encoding hVti1This studypFvM58hVti1 (amino acids 62–232) in yeast expression vector pVT100-U (2μ-URA3)This studypFvM60PCR-amplified hVti1 from λZAPII-hypothalamus cDNA in pBluescriptKS+This studypFvM61PCR-amplified hVti1 from λZAPII-hypothalamus cDNA in pBluescriptKS+This studypFvM91vti1–12 in pRS314 (CEN6-TRP1)This studypFvM92vti1–11in pRS314 (CEN6-TRP1)Fischer von Mollard et al. (25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar)pFvM93vti1–2 in pRS314 (CEN6-TRP1)Fischer von Mollard et al. (25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar)pFvM1042.9-kb EcoRI-SnaBI DNA from pRB58 with SUC2 in pRS316 (CEN6-URA3)Fischer von Mollard et al. (25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar)pFvM108vti1–11 with only the mutants E145G and L155F in pRS314 (CEN6-TRP1)This studypFvM1162.9-kbEcoRI-SnaBI DNA from pRB58 with SUC2in pRS315 (CEN6-LEU2)This studypFvM118hVti1 (amino acids 1–232) in yeast expression vector pVT100-U (2μ-URA3)This study Open table in a new tab hVti1 was PCR-amplified from a human glioblastoma cDNA library in pADANS (26Colicelli J. Birchmeier C. Michaeli T. O'Neill K. Riggs M. Wigler M. Proc. Natl. Acad. Sci U. S. A. 1989; 86: 3599-3603Crossref PubMed Scopus (163) Google Scholar) using an oligonucleotide complimentary to the ADH terminator (5′-AAC CTC TGG CGA AGA AGT CCA-3′) and complimentary to sequences 3′ of the hVti1-coding region (5′-CAG CCC ACA GCA ATA TGC-3′). The resulting 900-base pair fragment was gel-purified and cloned into EcoRV digested pBluescript KS+ to obtain pFvM46. To isolate pFvM60 and pFvM61, hVti1 was PCR-amplified from a human hypothalamus cDNA inserted into λZAPII (27Swaroop A. Xu J. Cytogenet. Cell Genet. 1993; 64: 292-294Crossref PubMed Scopus (22) Google Scholar) using a T7 oligonucleotide and the same 3′ hVti1 oligonucleotide, the fragment was gel purified, digested withSacI and XhoI, and cloned into pBluescript KS+. hVti1 (codons 62–232) was PCR-amplified from pFvM46 with the oligonucleotides 5′-CCG CTC GAG ATG GAG GAG GAG CTA C-3′ and 5′-CGG GAT CCT ACG CAT AGT CAG GAA CAT CAT ATG GGT AAT GGC TGC GAA AGA ATT TG-3′ and cloned into the multicopy yeast expression vector pVT100-U behind the ADH1 promoter (28Vernet T. Dignar D. Thomas D.Y. Gene (Amst.). 1987; 52: 225-233Crossref PubMed Scopus (465) Google Scholar) to obtain pFvM58. To construct pFvM118, hVti1 (codons 1–232) was PCR-amplified from pFvM50 using the oligonucleotide 5′-CCG CTC GAG ATG GCC TCC TCC GCC GCC TC-3′ and the same 3′ oligonucleotide as for pFvM58, and cloned into pVT100-U. pFvM91 encoding vti1-12 was generated by random PCR mutagenesis and was isolated in a screen for temperature sensitive growth defects in the same way as vti1-11 (25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar). The 2.8-kbScaI-BglII fragment from pFvM28 (encoding wild type VTI1) was ligated with the 3.9-kbScaI-BglII fragment of pFvM92 to construct pFvM108 (wild type N terminus codon 1–105, vti1-11 mutant C terminus codon 106–217). pFvM116 was constructed by subcloning a 2.9-kb EcoRI-SacI fragment from pRB58 (29Carlson M. Botstein D. Cell. 1982; 28: 145-154Abstract Full Text PDF PubMed Scopus (926) Google Scholar) encoding SUC2 into pRS315 (30Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). vti1-11 from pFvM108 was subcloned into the integration vector pRS306 (30Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). FvMY21 was constructed by integration of this plasmid linearized byXbaI into SEY6211 and looping out the wild typeVTI1 on 5-fluorourotic acid plates (31Boeke J.D. LaCroute F. Fink G.R. Mol. Gen. Genet. 1984; 197: 345-346Crossref PubMed Scopus (1712) Google Scholar). To identify human proteins that allow for growth in the absence of Vti1p, the strain FvMY6/pFvM16 (vti1Δ/pCEN-GAL1-VTI1) was used. FvMY6/pFvM16 cells were transformed with yeast expression plasmids encoding human proteins. This library consisted of human glioblastoma cDNAs fused to the ADH promoter and the first 14ADH1 codons in the multicopy yeast vector pADANS (26Colicelli J. Birchmeier C. Michaeli T. O'Neill K. Riggs M. Wigler M. Proc. Natl. Acad. Sci U. S. A. 1989; 86: 3599-3603Crossref PubMed Scopus (163) Google Scholar). Transformants were plated on SD-Leu plates, conditions that turn off the expression of VTI1 and prevent growth of FvMY6/pFvM16 cells because Vti1p is essential for growth. Colonies that grew were tested for the absence of Vti1p by immunoblot analysis. Plasmids were recovered and retransformed, and colonies that lost the pFvM16 plasmid were selected to confirm that suppression was dependent on the expression of a human protein. The insert of the suppressor plasmid pFvM50 was sequenced and encoded a human Vti1p homolog. The procedures for CPY and invertase immunoprecipitation were described earlier (25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar, 32Vater C.A. Raymond C.K. Ekena K. Howald S., I. Stevens T.H. J. Cell Biol. 1992; 119: 773-786Crossref PubMed Scopus (170) Google Scholar, 33Franzusoff A. Schekman R. EMBO J. 1989; 8: 2695-2702Crossref PubMed Scopus (170) Google Scholar). For CPY immunoprecipitations yeast cells were grown at 22 or 30 °C, radiolabeled with [35S]methionine for 10 min at the indicated temperature, and chased for 30 min after addition of 500 μg/ml methionine and cysteine. Invertase was derepressed by an incubation in minimal medium containing 0.1% glucose, 50 mm KPO4, pH 5.7, and 1 mg/ml bovine serum albumin for 30 min at 22 °C plus 15 min at 37 °C. Cells were radiolabeled for 7 min at 37 °C and chased for 0 min or 30 min. Cells were spheroplasted, and extracts were prepared from internal and external fractions, and CPY- or invertase-immunoprecipitated with polyclonal antibodies and fixedS. aureus cells. Immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A multicopy suppressor screen was used to identify human proteins that allow for survival of yeast cells in the absence of Vti1p. vti1Δcells expressing VTI1 under the control of the GAL1 promoter were transformed with the multicopy library pADANS. The pADANS library contains human glioblastoma cDNAs fused to the ADH1 promoter and the first 14 ADH1codons, which results in expression of human fusion proteins in yeast (26Colicelli J. Birchmeier C. Michaeli T. O'Neill K. Riggs M. Wigler M. Proc. Natl. Acad. Sci U. S. A. 1989; 86: 3599-3603Crossref PubMed Scopus (163) Google Scholar). In the resulting transformants, expression of VTI1 was turned off by plating the cells on media containing glucose. Colonies that grew were tested for the absence of Vti1p by immunoblot analysis. Plasmids were recovered from these suppressor strains, and retransformed into vti1Δ GAL-VTI1 cells. Colonies that lost the GAL1-VTI1 plasmid were selected to confirm that suppression was dependent on the expression of a human protein. Sequencing of the suppressor (pFvM50) revealed that the clone is predicted to encode a 232-amino acid protein with a C-terminal transmembrane domain. This protein displays 29% overall amino acid identity with the yeast Vti1p (25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar), and was therefore called hVti1 (Fig. 1 A). The 56-amino acid domain adjacent to the transmembrane domain is more homologous (41% amino acid identity). The regions between amino acid 38 and 67 and amino acid 160 and 193 are predicted to form amphipathic α-helical coiled coils using the paircoil program (probability score 0.71 and 0.69) (34Berger B. Wilson D.B. Wolf E. Tonchev T. Milla M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8259-8263Crossref PubMed Scopus (593) Google Scholar). The region in yeast Vti1p homologous to the second predicted coiled coil region is also predicted to form a coiled coil domain, indicating that the proteins may adopt similar structures. The first structural studies with SNARE proteins indicate that the isolated t-SNAREs SNAP-25 and Sec9p and the v-SNARE Snc1p are largely unstructured (35Rice L.M. Brennwald P. Brünger A.T. FEBS Lett. 1997; 415: 49-55Crossref PubMed Scopus (60) Google Scholar, 36Fasshauer D. Bruns D. Shen B. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 4582-4590Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). A large increase in α-helical contents was observed after formation of SNARE complexes. Complex formation may induce similar structural changes in Vti1p. By searching a data base of human expressed sequence tags (EST), human sequences that show high homology to the C-terminal half of the yeast Vti1p were identified (accession nos. N39768 and R68750). These sequences were identical to the C terminus of hVti1 identified in the suppressor screen. An oligonucleotide primer complimentary to sequences 3′ of the hVti1 and a primer containing vector sequences around the polylinker were used to PCR amplify the hVti1 from two different human cDNA libraries. One library consisted of human glioblastoma cDNA in the yeast expression plasmid pADANS (26Colicelli J. Birchmeier C. Michaeli T. O'Neill K. Riggs M. Wigler M. Proc. Natl. Acad. Sci U. S. A. 1989; 86: 3599-3603Crossref PubMed Scopus (163) Google Scholar). The other was human hypothalamus cDNA inserted into λZAPII phage (27Swaroop A. Xu J. Cytogenet. Cell Genet. 1993; 64: 292-294Crossref PubMed Scopus (22) Google Scholar). DNA sequences were amplified from both libraries, and these were predicted to encode a 171 amino acid protein starting at methionine 62 of hVti1 (Fig. 1 A, hVti1 starting at the arrowhead). A clone derived from the λ library (pFvM61) had an in-frame stop codon upstream of the putative initiating methionine 62. A different λ clone (pFvM60) and the clone derived from the pADANS library (pFvM 46) contained a coding region for the same 171-amino acid long hVti1 but were different upstream of the putative initiating methionine 62. pFvM46 ended after encoding 24 amino acids upstream of a putative initiating methionine which are present in hVti1, indicating that it is an incomplete clone. pFvM60 encoded 12 different amino acids upstream of methionine 62 (AMSDFRSVCRRQ) and did not contain an upstream in-frame stop codon. Northern blot analysis of human RNA from different tissues revealed that hVti1 was expressed as a single band of about 1.2 kb in all tissues (data not shown). This suggests that hVti1 has a role in basic cell function. Further data base searches revealed the presence of a Vti1-related hypothetical protein of unknown function in Schizosaccharomyces pombe, SpVti1 (Fig. 1 A), which shares 38% amino acid identity with S. cerevisiae Vti1p (Fig. 1 B). Recently, several Vti1p-related mouse ESTs were entered into the data base. The ESTs were assembled into a mouse Vti1 protein (mVti1), which is almost identical to hVti1 (93% amino acid identity). Surprisingly, a second Vti1-related protein, mVti1b, could be assembled from a second set of mouse ESTs. Unfortunately, the available sequences do not include the stop codon and end directly before the expected transmembrane domain. mVti1b shares only 31% amino acid identity with mVti1 and about the same degree of amino acid identity with yeast Vti1p (35%). Three human ESTs with high identities to mVti1b were found in the data base (AA326353, R29052, and T70362), indicating the presence of a hVti1b, but the sequence data are not extensive enough to assemble the full protein. In Vti1p the domain between amino acid 37 and 60 is predicted to form an amphipathic α-helix which contains charged amino acids on one side and the other face is strongly hydrophobic due to the presence of bulky hydrophobic amino acids (Fig. 2 A). The homologous domains in the other Vti1p-like proteins (Fig. 1, between filled circles) also display similar properties. In hVti1 and mVti1 the hydrophobic face is less pronounced but the domain between amino acid residues 77 and 98 can also form an amphipathic α-helix. These domains resemble amphipathic α-helical fusion peptides found in viral fusion proteins and are also present close to the N terminus in other v-SNAREs (37White J.M. Science. 1992; 258: 917-924Crossref PubMed Scopus (653) Google Scholar, 38Dascher C. Ossig R. Gallwitz D. Schmitt H.D. Mol. Cell. Biol. 1991; 11: 872-885Crossref PubMed Scopus (280) Google Scholar, 39Jahn R. Südhof T.C. Annu. Rev. Neurosci. 1994; 17: 219-246Crossref PubMed Scopus (341) Google Scholar). An alignment of all Vti1 proteins revealed a domain of 75 amino acids next to the transmembrane domain, which could be aligned without gaps and shows blocks of high amino acid identity. Within this domain all Vti1 proteins contain amino acid stretches that are predicted to form amphipathic α-helical coiled coils with high probability (Fig. 1, solid lines above the sequences) and lower probability (dashed lines). Using the paircoil program (34Berger B. Wilson D.B. Wolf E. Tonchev T. Milla M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8259-8263Crossref PubMed Scopus (593) Google Scholar) the probability scores were: hVti1 0.69, mVti1 0.74, mVti1b 0.30, SpVti1 0.85, and Vti1 0.29. Coiled coil formation may require interaction with other proteins as observed for SNAP-25, Sec9p, and the v-SNARE Snc1p (35Rice L.M. Brennwald P. Brünger A.T. FEBS Lett. 1997; 415: 49-55Crossref PubMed Scopus (60) Google Scholar, 36Fasshauer D. Bruns D. Shen B. Jahn R. Brünger A.T. J. Biol. Chem. 1997; 272: 4582-4590Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). To obtain a reliable secondary structure prediction all Vti1 proteins were aligned using the Maxhom multiple sequence alignment program (40Sander C. Schneider R. Proteins. 1991; 9: 56-68Crossref PubMed Scopus (1489) Google Scholar), and the secondary structure of the alignment predicted with the EMBL PHDsec program (41Rost B. Sander C. J. Mol. Biol. 1993; 232: 584-599Crossref PubMed Scopus (2656) Google Scholar). The domain between amino acids 132 and 190 in the Vti1 alignment is predicted to be α-helical with a very high probability. To visualize conserved features, the Vti1 alignment between amino acids 134 and 187 (Fig. 1 A, solid squares) was drawn as a helical wheel projection (Fig. 2 B). Amino acid residues that were identical in all Vti1 proteins were depicted in uppercase letters. Residues that are identical in three out of the four Vti1 proteins were drawn aslowercase letters. hVti1 and mVti1 were treated as one protein due to their high degree of identity. Bulky hydrophobic amino acids (L, M, V, I) are boxed, an empty boxrepresents a conserved bulky hydrophobic residue. Charged residues are surrounded by a circle, a circle with a dash (–) represents a conserved E or D, a circle with a plus (+) represents a conserved K or R. X marks residues that are not conserved. The helical wheel projection reveals that one face of the helix is highly conserved. It consists of three leucines, an alanine, a glycine, and three more leucines. Only in two cases is one of the leucines replaced by an isoleucine. The neighboring face of the helix contains a conserved hydrophobic and conserved charged amino acids, two identical arginines, an acidic residue, and a less conserved basic residue. The other parts of the helix are less well conserved. As described recently, screens for the temperature-sensitivevti1 mutants in yeast led to the identification of two different classes of mutants (25Fischer von Mollard G. Nothwehr S.F. Stevens T.H. J. Cell Biol. 1997; 137: 1511-1524Crossref PubMed Scopus (175) Google Scholar). The mutants vti1-1 and vti1-2 exhibit a block in transport of the vacuolar hydrolase CPY from the late Golgi to the prevacuolar compartment at the restrictive temperature. The mutant vti1-11 has a temperature-sensitive growth defect and accumulates secretory proteins in the ER and early Golgi compartment, in addition to a block in Golgi to prevacuolar traffic. To determine which parts of Vti1p are involved in these functions the vti1 mutant alleles were sequenced. vti1-1 contains the amino acid exchanges E145K and G148R. vti1-2 has the mutations S130P and I151T. 8 amino acid exchanges were identified in vti1-11 (Y8R, K20R, H40R, N61S, K73R, Q84R, E145G, and L155F). The construction of a hybrid protein encoded by the plasmid pFvM108 revealed that yeast cells carrying a protein with only the amino acid exchanges E145G and L155F exhibited phenotypes identical to the original vti1-11 mutant (data not shown). Therefore E145G and L155F are responsible for the trafficking defect observed for vti1-11. In our ongoing analysis of yeast Vti1p function we analyzed a newVTI1 allele, vti1-12. The fate of newly synthesized CPY was monitored in vti1-12 cells incubated at 22 °C or for 15 min at 37 °C by pulse-chase labeling followed by CPY immunoprecipitation. Even at 22 °C vti1-12 cells accumulated a significant proportion of CPY in the ER and early Golgi forms (p1CPY) and the late Golgi form p2CPY intracellularly (42Stevens T. Esmon B. Schekman R. Cell. 1982; 30: 439-448Abstract Full Text PDF PubMed Scopus (371) Google Scholar). Hardly any vacuolar localized mature CPY (mCPY) was present (Fig. 3, left panel, I). These cells also secreted p2CPY (Fig. 3, left panel, E). At 37 °C vti1-12 cells exhibited a severe growth defect and accumulated an even higher proportion of CPY in the ER and early Golgi form (Fig. 3, right panel). These data indicate that the vti1-12 mutation causes a constitutive block of traffic from the late Golgi to the vacuole and a temperature-sensitive block in traffic to the cis-