Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Mol Cell Biol. 1991 Feb; 11(2): 872–885.
PMCID: PMC359739
PMID: 1990290

Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the RAS superfamily.

Abstract

In Saccharomyces cerevisiae, the GTP-binding Ypt1 protein (Ypt1p) is essential for endoplasmic reticulum-to-Golgi protein transport. By exploiting a GAL10-YPT1 fusion to regulate YPT1 expression, three multicopy suppressors, SLY2, SLY12, and SLY41, and a single-copy suppressor, SLY1-20, that allowed YPT1-independent growth were isolated. Wild-type Sly1p is hydrophilic, is essential for cell viability, and differs from Sly1-20p by a single amino acid. SLY2 and SLY12 encode proteins with hydrophobic tails similar to synaptobrevins, integral membrane proteins of synaptic vesicles in higher eucaryotes. Sly41p is hydrophobic and exhibits sequence similarities with the chloroplast phosphate translocator. SLY12 but not SLY41 is an essential gene. The SLY2 null mutant is cold and heat sensitive. The SLY gene products may comprise elements of the protein transport machinery.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (3.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  • Achstetter T, Franzusoff A, Field C, Schekman R. SEC7 encodes an unusual, high molecular weight protein required for membrane traffic from the yeast Golgi apparatus. J Biol Chem. 1988 Aug 25;263(24):11711–11717. [PubMed] [Google Scholar]
  • Argos P. A sensitive procedure to compare amino acid sequences. J Mol Biol. 1987 Jan 20;193(2):385–396. [PubMed] [Google Scholar]
  • Bacon RA, Salminen A, Ruohola H, Novick P, Ferro-Novick S. The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants. J Cell Biol. 1989 Sep;109(3):1015–1022. [PMC free article] [PubMed] [Google Scholar]
  • Baker D, Wuestehube L, Schekman R, Botstein D, Segev N. GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction. Proc Natl Acad Sci U S A. 1990 Jan;87(1):355–359. [PMC free article] [PubMed] [Google Scholar]
  • Bankaitis VA, Malehorn DE, Emr SD, Greene R. The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J Cell Biol. 1989 Apr;108(4):1271–1281. [PMC free article] [PubMed] [Google Scholar]
  • Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. [PubMed] [Google Scholar]
  • Bender A, Pringle JR. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9976–9980. [PMC free article] [PubMed] [Google Scholar]
  • Bernstein M, Hoffmann W, Ammerer G, Schekman R. Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum. J Cell Biol. 1985 Dec;101(6):2374–2382. [PMC free article] [PubMed] [Google Scholar]
  • Bernstein M, Kepes F, Schekman R. Sec59 encodes a membrane protein required for core glycosylation in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Mar;9(3):1191–1199. [PMC free article] [PubMed] [Google Scholar]
  • Böhni PC, Deshaies RJ, Schekman RW. SEC11 is required for signal peptide processing and yeast cell growth. J Cell Biol. 1988 Apr;106(4):1035–1042. [PMC free article] [PubMed] [Google Scholar]
  • Bourne HR. Do GTPases direct membrane traffic in secretion? Cell. 1988 Jun 3;53(5):669–671. [PubMed] [Google Scholar]
  • Broach JR, Strathern JN, Hicks JB. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. [PubMed] [Google Scholar]
  • Broek D, Toda T, Michaeli T, Levin L, Birchmeier C, Zoller M, Powers S, Wigler M. The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway. Cell. 1987 Mar 13;48(5):789–799. [PubMed] [Google Scholar]
  • Cannon JF, Gibbs JB, Tatchell K. Suppressors of the ras2 mutation of Saccharomyces cerevisiae. Genetics. 1986 Jun;113(2):247–264. [PMC free article] [PubMed] [Google Scholar]
  • Carlson M, Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. [PubMed] [Google Scholar]
  • Chardin P. The ras superfamily proteins. Biochimie. 1988 Jul;70(7):865–868. [PubMed] [Google Scholar]
  • Chen EY, Seeburg PH. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. [PubMed] [Google Scholar]
  • Eakle KA, Bernstein M, Emr SD. Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product. Mol Cell Biol. 1988 Oct;8(10):4098–4109. [PMC free article] [PubMed] [Google Scholar]
  • Flügge UI, Fischer K, Gross A, Sebald W, Lottspeich F, Eckerskorn C. The triose phosphate-3-phosphoglycerate-phosphate translocator from spinach chloroplasts: nucleotide sequence of a full-length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts. EMBO J. 1989 Jan;8(1):39–46. [PMC free article] [PubMed] [Google Scholar]
  • Gallwitz D, Donath C, Sander C. A yeast gene encoding a protein homologous to the human c-has/bas proto-oncogene product. Nature. 1983 Dec 15;306(5944):704–707. [PubMed] [Google Scholar]
  • Garnier J, Osguthorpe DJ, Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. [PubMed] [Google Scholar]
  • Garrett MD, Self AJ, van Oers C, Hall A. Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins. J Biol Chem. 1989 Jan 5;264(1):10–13. [PubMed] [Google Scholar]
  • Goud B, Salminen A, Walworth NC, Novick PJ. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell. 1988 Jun 3;53(5):753–768. [PubMed] [Google Scholar]
  • Haubruck H, Disela C, Wagner P, Gallwitz D. The ras-related ypt protein is an ubiquitous eukaryotic protein: isolation and sequence analysis of mouse cDNA clones highly homologous to the yeast YPT1 gene. EMBO J. 1987 Dec 20;6(13):4049–4053. [PMC free article] [PubMed] [Google Scholar]
  • Haubruck H, Prange R, Vorgias C, Gallwitz D. The ras-related mouse ypt1 protein can functionally replace the YPT1 gene product in yeast. EMBO J. 1989 May;8(5):1427–1432. [PMC free article] [PubMed] [Google Scholar]
  • Hicke L, Schekman R. Yeast Sec23p acts in the cytoplasm to promote protein transport from the endoplasmic reticulum to the Golgi complex in vivo and in vitro. EMBO J. 1989 Jun;8(6):1677–1684. [PMC free article] [PubMed] [Google Scholar]
  • Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. [PMC free article] [PubMed] [Google Scholar]
  • Jähnig F. Structure predictions of membrane proteins are not that bad. Trends Biochem Sci. 1990 Mar;15(3):93–95. [PubMed] [Google Scholar]
  • Johnston SA, Hopper JE. Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6971–6975. [PMC free article] [PubMed] [Google Scholar]
  • Kikuchi A, Sasaki T, Araki S, Hata Y, Takai Y. Purification and characterization from bovine brain cytosol of two GTPase-activating proteins specific for smg p21, a GTP-binding protein having the same effector domain as c-ras p21s. J Biol Chem. 1989 Jun 5;264(16):9133–9136. [PubMed] [Google Scholar]
  • Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. [PubMed] [Google Scholar]
  • Landschulz WH, Johnson PF, McKnight SL. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. [PubMed] [Google Scholar]
  • Langford CJ, Gallwitz D. Evidence for an intron-contained sequence required for the splicing of yeast RNA polymerase II transcripts. Cell. 1983 Jun;33(2):519–527. [PubMed] [Google Scholar]
  • Langford CJ, Klinz FJ, Donath C, Gallwitz D. Point mutations identify the conserved, intron-contained TACTAAC box as an essential splicing signal sequence in yeast. Cell. 1984 Mar;36(3):645–653. [PubMed] [Google Scholar]
  • Laughon A, Gesteland RF. Primary structure of the Saccharomyces cerevisiae GAL4 gene. Mol Cell Biol. 1984 Feb;4(2):260–267. [PMC free article] [PubMed] [Google Scholar]
  • Leer RJ, van Raamsdonk-Duin MM, Molenaar CM, Witsenboer HM, Mager WH, Planta RJ. Yeast contains two functional genes coding for ribosomal protein S10. Nucleic Acids Res. 1985 Jul 25;13(14):5027–5039. [PMC free article] [PubMed] [Google Scholar]
  • Matsumoto K, Toh-e A, Oshima Y. Genetic control of galactokinase synthesis in Saccharomyces cerevisiae: evidence for constitutive expression of the positive regulatory gene gal4. J Bacteriol. 1978 May;134(2):446–457. [PMC free article] [PubMed] [Google Scholar]
  • Matsumoto K, Uno I, Oshima Y, Ishikawa T. Isolation and characterization of yeast mutants deficient in adenylate cyclase and cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2355–2359. [PMC free article] [PubMed] [Google Scholar]
  • Milburn MV, Tong L, deVos AM, Brünger A, Yamaizumi Z, Nishimura S, Kim SH. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science. 1990 Feb 23;247(4945):939–945. [PubMed] [Google Scholar]
  • Molenaar CM, Prange R, Gallwitz D. A carboxyl-terminal cysteine residue is required for palmitic acid binding and biological activity of the ras-related yeast YPT1 protein. EMBO J. 1988 Apr;7(4):971–976. [PMC free article] [PubMed] [Google Scholar]
  • Murray AW, Szostak JW. Pedigree analysis of plasmid segregation in yeast. Cell. 1983 Oct;34(3):961–970. [PubMed] [Google Scholar]
  • Nair J, Müller H, Peterson M, Novick P. Sec2 protein contains a coiled-coil domain essential for vesicular transport and a dispensable carboxy terminal domain. J Cell Biol. 1990 Jun;110(6):1897–1909. [PMC free article] [PubMed] [Google Scholar]
  • Nakano A, Brada D, Schekman R. A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast. J Cell Biol. 1988 Sep;107(3):851–863. [PMC free article] [PubMed] [Google Scholar]
  • Nakańo A, Muramatsu M. A novel GTP-binding protein, Sar1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Biol. 1989 Dec;109(6 Pt 1):2677–2691. [PMC free article] [PubMed] [Google Scholar]
  • Newman AP, Shim J, Ferro-Novick S. BET1, BOS1, and SEC22 are members of a group of interacting yeast genes required for transport from the endoplasmic reticulum to the Golgi complex. Mol Cell Biol. 1990 Jul;10(7):3405–3414. [PMC free article] [PubMed] [Google Scholar]
  • Novick P, Ferro S, Schekman R. Order of events in the yeast secretory pathway. Cell. 1981 Aug;25(2):461–469. [PubMed] [Google Scholar]
  • Pai EF, Kabsch W, Krengel U, Holmes KC, John J, Wittinghofer A. Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature. 1989 Sep 21;341(6239):209–214. [PubMed] [Google Scholar]
  • Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. [PMC free article] [PubMed] [Google Scholar]
  • Rose MD, Novick P, Thomas JH, Botstein D, Fink GR. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. [PubMed] [Google Scholar]
  • Rothstein RJ. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. [PubMed] [Google Scholar]
  • Rudolph HK, Antebi A, Fink GR, Buckley CM, Dorman TE, LeVitre J, Davidow LS, Mao JI, Moir DT. The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family. Cell. 1989 Jul 14;58(1):133–145. [PubMed] [Google Scholar]
  • Ruggieri R, Tanaka K, Nakafuku M, Kaziro Y, Toh-e A, Matsumoto K. MSI1, a negative regulator of the RAS-cAMP pathway in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8778–8782. [PMC free article] [PubMed] [Google Scholar]
  • Salminen A, Novick PJ. The Sec15 protein responds to the function of the GTP binding protein, Sec4, to control vesicular traffic in yeast. J Cell Biol. 1989 Sep;109(3):1023–1036. [PMC free article] [PubMed] [Google Scholar]
  • Salminen A, Novick PJ. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. [PubMed] [Google Scholar]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed] [Google Scholar]
  • Schmitt HD, Puzicha M, Gallwitz D. Study of a temperature-sensitive mutant of the ras-related YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium. Cell. 1988 May 20;53(4):635–647. [PubMed] [Google Scholar]
  • Schmitt HD, Wagner P, Pfaff E, Gallwitz D. The ras-related YPT1 gene product in yeast: a GTP-binding protein that might be involved in microtubule organization. Cell. 1986 Nov 7;47(3):401–412. [PubMed] [Google Scholar]
  • Segev N, Botstein D. The ras-like yeast YPT1 gene is itself essential for growth, sporulation, and starvation response. Mol Cell Biol. 1987 Jul;7(7):2367–2377. [PMC free article] [PubMed] [Google Scholar]
  • Segev N, Mulholland J, Botstein D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 1988 Mar 25;52(6):915–924. [PubMed] [Google Scholar]
  • Stearns T, Willingham MC, Botstein D, Kahn RA. ADP-ribosylation factor is functionally and physically associated with the Golgi complex. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1238–1242. [PMC free article] [PubMed] [Google Scholar]
  • Struhl K. Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region. Nucleic Acids Res. 1985 Dec 9;13(23):8587–8601. [PMC free article] [PubMed] [Google Scholar]
  • Südhof TC, Baumert M, Perin MS, Jahn R. A synaptic vesicle membrane protein is conserved from mammals to Drosophila. Neuron. 1989 May;2(5):1475–1481. [PubMed] [Google Scholar]
  • Szostak JW, Orr-Weaver TL, Rothstein RJ, Stahl FW. The double-strand-break repair model for recombination. Cell. 1983 May;33(1):25–35. [PubMed] [Google Scholar]
  • Tanaka K, Matsumoto K, Toh-E A. IRA1, an inhibitory regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):757–768. [PMC free article] [PubMed] [Google Scholar]
  • Tautz D, Renz M. An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem. 1983 Jul 1;132(1):14–19. [PubMed] [Google Scholar]
  • Thompson RC. EFTu provides an internal kinetic standard for translational accuracy. Trends Biochem Sci. 1988 Mar;13(3):91–93. [PubMed] [Google Scholar]
  • Toda T, Cameron S, Sass P, Zoller M, Wigler M. Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell. 1987 Jul 17;50(2):277–287. [PubMed] [Google Scholar]
  • Toda T, Uno I, Ishikawa T, Powers S, Kataoka T, Broek D, Cameron S, Broach J, Matsumoto K, Wigler M. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell. 1985 Jan;40(1):27–36. [PubMed] [Google Scholar]
  • Touchot N, Chardin P, Tavitian A. Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8210–8214. [PMC free article] [PubMed] [Google Scholar]
  • Trahey M, Wong G, Halenbeck R, Rubinfeld B, Martin GA, Ladner M, Long CM, Crosier WJ, Watt K, Koths K, et al. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. [PubMed] [Google Scholar]
  • Trimble WS, Cowan DM, Scheller RH. VAMP-1: a synaptic vesicle-associated integral membrane protein. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4538–4542. [PMC free article] [PubMed] [Google Scholar]
  • Vogel US, Dixon RA, Schaber MD, Diehl RE, Marshall MS, Scolnick EM, Sigal IS, Gibbs JB. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. [PubMed] [Google Scholar]
  • Walworth NC, Goud B, Kabcenell AK, Novick PJ. Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic. EMBO J. 1989 Jun;8(6):1685–1693. [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

-