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. 1999 Aug;152(4):1543–1556. doi: 10.1093/genetics/152.4.1543

Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers.

S Jones 1, G Jedd 1, R A Kahn 1, A Franzusoff 1, F Bartolini 1, N Segev 1
PMCID: PMC1460709  PMID: 10430582

Abstract

Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport. While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment. Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well. Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation. Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases. Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function. Sec7p itself has an important role in the yeast secretory pathway. However, its mechanism of action is not yet understood. We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes. Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt GTPases. The Sec7-domain of Sec7p is sufficient for this activity. Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a fungal metabolite that inhibits some of the Arf-GEFs, indicating that this domain is a target for BFA. These results demonstrate that the ability to act as Arf-GEFs is a general property of all Sec7-domain proteins in yeast. The genetic interactions observed between Arf GEFs and Ypt GTPases suggest the existence of a Ypt-Arf GTPase cascade in the secretory pathway.

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Selected References

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  1. 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]
  2. Bacon R. A., 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. doi: 10.1083/jcb.109.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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. doi: 10.1073/pnas.87.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Betz S. F., Schnuchel A., Wang H., Olejniczak E. T., Meadows R. P., Lipsky B. P., Harris E. A., Staunton D. E., Fesik S. W. Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1. Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):7909–7914. doi: 10.1073/pnas.95.14.7909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boman A. L., Taylor T. C., Melançon P., Wilson K. L. A role for ADP-ribosylation factor in nuclear vesicle dynamics. Nature. 1992 Aug 6;358(6386):512–514. doi: 10.1038/358512a0. [DOI] [PubMed] [Google Scholar]
  6. 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. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  7. Caumont A. S., Galas M. C., Vitale N., Aunis D., Bader M. F. Regulated exocytosis in chromaffin cells. Translocation of ARF6 stimulates a plasma membrane-associated phospholipase D. J Biol Chem. 1998 Jan 16;273(3):1373–1379. doi: 10.1074/jbc.273.3.1373. [DOI] [PubMed] [Google Scholar]
  8. Chardin P., Paris S., Antonny B., Robineau S., Béraud-Dufour S., Jackson C. L., Chabre M. A human exchange factor for ARF contains Sec7- and pleckstrin-homology domains. Nature. 1996 Dec 5;384(6608):481–484. doi: 10.1038/384481a0. [DOI] [PubMed] [Google Scholar]
  9. Cherfils J., Ménétrey J., Mathieu M., Le Bras G., Robineau S., Béraud-Dufour S., Antonny B., Chardin P. Structure of the Sec7 domain of the Arf exchange factor ARNO. Nature. 1998 Mar 5;392(6671):101–105. doi: 10.1038/32210. [DOI] [PubMed] [Google Scholar]
  10. D'Souza-Schorey C., van Donselaar E., Hsu V. W., Yang C., Stahl P. D., Peters P. J. ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation. J Cell Biol. 1998 Feb 9;140(3):603–616. doi: 10.1083/jcb.140.3.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dascher C., Balch W. E. Dominant inhibitory mutants of ARF1 block endoplasmic reticulum to Golgi transport and trigger disassembly of the Golgi apparatus. J Biol Chem. 1994 Jan 14;269(2):1437–1448. [PubMed] [Google Scholar]
  12. Deitz S. B., Wu C., Silve S., Howell K. E., Melançon P., Kahn R. A., Franzusoff A. Human ARF4 expression rescues sec7 mutant yeast cells. Mol Cell Biol. 1996 Jul;16(7):3275–3284. doi: 10.1128/mcb.16.7.3275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Donaldson J. G., Finazzi D., Klausner R. D. Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein. Nature. 1992 Nov 26;360(6402):350–352. doi: 10.1038/360350a0. [DOI] [PubMed] [Google Scholar]
  14. Enomoto S., Chen G., Berman J. Vectors for expressing T7 epitope- and His6 affinity-tagged fusion proteins in S. cerevisiae. Biotechniques. 1998 May;24(5):782-6, 788. doi: 10.2144/98245st01. [DOI] [PubMed] [Google Scholar]
  15. Esmon B., Novick P., Schekman R. Compartmentalized assembly of oligosaccharides on exported glycoproteins in yeast. Cell. 1981 Aug;25(2):451–460. doi: 10.1016/0092-8674(81)90063-5. [DOI] [PubMed] [Google Scholar]
  16. Ferro-Novick S., Jahn R. Vesicle fusion from yeast to man. Nature. 1994 Jul 21;370(6486):191–193. doi: 10.1038/370191a0. [DOI] [PubMed] [Google Scholar]
  17. Franco M., Chardin P., Chabre M., Paris S. Myristoylation-facilitated binding of the G protein ARF1GDP to membrane phospholipids is required for its activation by a soluble nucleotide exchange factor. J Biol Chem. 1996 Jan 19;271(3):1573–1578. doi: 10.1074/jbc.271.3.1573. [DOI] [PubMed] [Google Scholar]
  18. Frank S., Upender S., Hansen S. H., Casanova J. E. ARNO is a guanine nucleotide exchange factor for ADP-ribosylation factor 6. J Biol Chem. 1998 Jan 2;273(1):23–27. doi: 10.1074/jbc.273.1.23. [DOI] [PubMed] [Google Scholar]
  19. Gaynor E. C., Chen C. Y., Emr S. D., Graham T. R. ARF is required for maintenance of yeast Golgi and endosome structure and function. Mol Biol Cell. 1998 Mar;9(3):653–670. doi: 10.1091/mbc.9.3.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Goud B., Salminen A., Walworth N. C., Novick P. J. 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. doi: 10.1016/0092-8674(88)90093-1. [DOI] [PubMed] [Google Scholar]
  22. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  23. Jedd G., Mulholland J., Segev N. Two new Ypt GTPases are required for exit from the yeast trans-Golgi compartment. J Cell Biol. 1997 May 5;137(3):563–580. doi: 10.1083/jcb.137.3.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jedd G., Richardson C., Litt R., Segev N. The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway. J Cell Biol. 1995 Nov;131(3):583–590. doi: 10.1083/jcb.131.3.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jones S., Litt R. J., Richardson C. J., Segev N. Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport. J Cell Biol. 1995 Sep;130(5):1051–1061. doi: 10.1083/jcb.130.5.1051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kahn R. A., Clark J., Rulka C., Stearns T., Zhang C. J., Randazzo P. A., Terui T., Cavenagh M. Mutational analysis of Saccharomyces cerevisiae ARF1. J Biol Chem. 1995 Jan 6;270(1):143–150. doi: 10.1074/jbc.270.1.143. [DOI] [PubMed] [Google Scholar]
  27. Kaiser C. A., Schekman R. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell. 1990 May 18;61(4):723–733. doi: 10.1016/0092-8674(90)90483-u. [DOI] [PubMed] [Google Scholar]
  28. Klarlund J. K., Rameh L. E., Cantley L. C., Buxton J. M., Holik J. J., Sakelis C., Patki V., Corvera S., Czech M. P. Regulation of GRP1-catalyzed ADP ribosylation factor guanine nucleotide exchange by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1998 Jan 23;273(4):1859–1862. doi: 10.1074/jbc.273.4.1859. [DOI] [PubMed] [Google Scholar]
  29. Klausner R. D., Donaldson J. G., Lippincott-Schwartz J. Brefeldin A: insights into the control of membrane traffic and organelle structure. J Cell Biol. 1992 Mar;116(5):1071–1080. doi: 10.1083/jcb.116.5.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ktistakis N. T., Brown H. A., Waters M. G., Sternweis P. C., Roth M. G. Evidence that phospholipase D mediates ADP ribosylation factor-dependent formation of Golgi coated vesicles. J Cell Biol. 1996 Jul;134(2):295–306. doi: 10.1083/jcb.134.2.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lazar T., Götte M., Gallwitz D. Vesicular transport: how many Ypt/Rab-GTPases make a eukaryotic cell? Trends Biochem Sci. 1997 Dec;22(12):468–472. doi: 10.1016/s0968-0004(97)01150-x. [DOI] [PubMed] [Google Scholar]
  32. Lee F. J., Stevens L. A., Kao Y. L., Moss J., Vaughan M. Characterization of a glucose-repressible ADP-ribosylation factor 3 (ARF3) from Saccharomyces cerevisiae. J Biol Chem. 1994 Aug 19;269(33):20931–20937. [PubMed] [Google Scholar]
  33. Lupashin V. V., Hamamoto S., Schekman R. W. Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes. J Cell Biol. 1996 Feb;132(3):277–289. doi: 10.1083/jcb.132.3.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meacci E., Tsai S. C., Adamik R., Moss J., Vaughan M. Cytohesin-1, a cytosolic guanine nucleotide-exchange protein for ADP-ribosylation factor. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1745–1748. doi: 10.1073/pnas.94.5.1745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Morinaga N., Moss J., Vaughan M. Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12926–12931. doi: 10.1073/pnas.94.24.12926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mossessova E., Gulbis J. M., Goldberg J. Structure of the guanine nucleotide exchange factor Sec7 domain of human arno and analysis of the interaction with ARF GTPase. Cell. 1998 Feb 6;92(3):415–423. doi: 10.1016/s0092-8674(00)80933-2. [DOI] [PubMed] [Google Scholar]
  37. Novick P., Zerial M. The diversity of Rab proteins in vesicle transport. Curr Opin Cell Biol. 1997 Aug;9(4):496–504. doi: 10.1016/s0955-0674(97)80025-7. [DOI] [PubMed] [Google Scholar]
  38. Orci L., Tagaya M., Amherdt M., Perrelet A., Donaldson J. G., Lippincott-Schwartz J., Klausner R. D., Rothman J. E. Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae. Cell. 1991 Mar 22;64(6):1183–1195. doi: 10.1016/0092-8674(91)90273-2. [DOI] [PubMed] [Google Scholar]
  39. Peyroche A., Paris S., Jackson C. L. Nucleotide exchange on ARF mediated by yeast Gea1 protein. Nature. 1996 Dec 5;384(6608):479–481. doi: 10.1038/384479a0. [DOI] [PubMed] [Google Scholar]
  40. Randazzo P. A., Weiss O., Kahn R. A. Preparation of recombinant ADP-ribosylation factor. Methods Enzymol. 1995;257:128–135. doi: 10.1016/s0076-6879(95)57018-7. [DOI] [PubMed] [Google Scholar]
  41. Rexach M. F., Schekman R. W. Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles. J Cell Biol. 1991 Jul;114(2):219–229. doi: 10.1083/jcb.114.2.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rothman J. E., Wieland F. T. Protein sorting by transport vesicles. Science. 1996 Apr 12;272(5259):227–234. doi: 10.1126/science.272.5259.227. [DOI] [PubMed] [Google Scholar]
  43. Sata M., Donaldson J. G., Moss J., Vaughan M. Brefeldin A-inhibited guanine nucleotide-exchange activity of Sec7 domain from yeast Sec7 with yeast and mammalian ADP ribosylation factors. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4204–4208. doi: 10.1073/pnas.95.8.4204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schekman R., Orci L. Coat proteins and vesicle budding. Science. 1996 Mar 15;271(5255):1526–1533. doi: 10.1126/science.271.5255.1526. [DOI] [PubMed] [Google Scholar]
  45. Schuler G. D., Altschul S. F., Lipman D. J. A workbench for multiple alignment construction and analysis. Proteins. 1991;9(3):180–190. doi: 10.1002/prot.340090304. [DOI] [PubMed] [Google Scholar]
  46. 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. doi: 10.1016/0092-8674(88)90433-3. [DOI] [PubMed] [Google Scholar]
  47. Sewell J. L., Kahn R. A. Sequences of the bovine and yeast ADP-ribosylation factor and comparison to other GTP-binding proteins. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4620–4624. doi: 10.1073/pnas.85.13.4620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Stearns T., Willingham M. C., Botstein D., Kahn R. A. 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. doi: 10.1073/pnas.87.3.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stevens T., Esmon B., Schekman R. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell. 1982 Sep;30(2):439–448. doi: 10.1016/0092-8674(82)90241-0. [DOI] [PubMed] [Google Scholar]
  50. Taylor T. C., Kahn R. A., Melançon P. Two distinct members of the ADP-ribosylation factor family of GTP-binding proteins regulate cell-free intra-Golgi transport. Cell. 1992 Jul 10;70(1):69–79. doi: 10.1016/0092-8674(92)90534-j. [DOI] [PubMed] [Google Scholar]
  51. Wach A., Brachat A., Pöhlmann R., Philippsen P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 1994 Dec;10(13):1793–1808. doi: 10.1002/yea.320101310. [DOI] [PubMed] [Google Scholar]
  52. Wolf J., Nicks M., Deitz S., van Tuinen E., Franzusoff A. An N-end rule destabilization mutant reveals pre-Golgi requirements for Sec7p in yeast membrane traffic. Biochem Biophys Res Commun. 1998 Feb 4;243(1):191–198. doi: 10.1006/bbrc.1998.8084. [DOI] [PubMed] [Google Scholar]
  53. Zhang C. J., Rosenwald A. G., Willingham M. C., Skuntz S., Clark J., Kahn R. A. Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo. J Cell Biol. 1994 Feb;124(3):289–300. doi: 10.1083/jcb.124.3.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhao L., Helms J. B., Brügger B., Harter C., Martoglio B., Graf R., Brunner J., Wieland F. T. Direct and GTP-dependent interaction of ADP ribosylation factor 1 with coatomer subunit beta. Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4418–4423. doi: 10.1073/pnas.94.9.4418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Zheng Y., Bender A., Cerione R. A. Interactions among proteins involved in bud-site selection and bud-site assembly in Saccharomyces cerevisiae. J Biol Chem. 1995 Jan 13;270(2):626–630. doi: 10.1074/jbc.270.2.626. [DOI] [PubMed] [Google Scholar]
  56. Zheng Y., Cerione R., Bender A. Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3. J Biol Chem. 1994 Jan 28;269(4):2369–2372. [PubMed] [Google Scholar]

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