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. 1990 Aug;9(8):2375–2383. doi: 10.1002/j.1460-2075.1990.tb07412.x

Synthetic peptides of the Rab effector domain inhibit vesicular transport through the secretory pathway.

H Plutner 1, R Schwaninger 1, S Pind 1, W E Balch 1
PMCID: PMC552261  PMID: 2114975

Abstract

Synthetic peptides of the putative effector domain of members of the ras-related rab gene family of small GTP-binding proteins were synthesized and found to be potent inhibitors of endoplasmic reticulum (ER) to Golgi and intra-Golgi transport in vitro. Inhibition of transport by one of the effector domain peptides was rapid (t1/2 of 30 s), and irreversible. Analysis of the temporal site of peptide inhibition indicated that a late step in transport was blocked, coincident with a Ca2(+)-dependent prefusion step. The results provide novel biochemical evidence for the role of members of the rab gene family in vesicular transport in mammalian cells, and implicate a role for a new downstream Rab effector protein (REP) regulating vesicle fusion.

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Selected References

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

  1. Adari H., Lowy D. R., Willumsen B. M., Der C. J., McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. doi: 10.1126/science.2833817. [DOI] [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., Hicke L., Rexach M., Schleyer M., Schekman R. Reconstitution of SEC gene product-dependent intercompartmental protein transport. Cell. 1988 Jul 29;54(3):335–344. doi: 10.1016/0092-8674(88)90196-1. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Balch W. E. Biochemistry of interorganelle transport. A new frontier in enzymology emerges from versatile in vitro model systems. J Biol Chem. 1989 Oct 15;264(29):16965–16968. [PubMed] [Google Scholar]
  6. Balch W. E., Dunphy W. G., Braell W. A., Rothman J. E. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N-acetylglucosamine. Cell. 1984 Dec;39(2 Pt 1):405–416. doi: 10.1016/0092-8674(84)90019-9. [DOI] [PubMed] [Google Scholar]
  7. Balch W. E., Glick B. S., Rothman J. E. Sequential intermediates in the pathway of intercompartmental transport in a cell-free system. Cell. 1984 Dec;39(3 Pt 2):525–536. doi: 10.1016/0092-8674(84)90459-8. [DOI] [PubMed] [Google Scholar]
  8. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  9. Beckers C. J., Balch W. E. Calcium and GTP: essential components in vesicular trafficking between the endoplasmic reticulum and Golgi apparatus. J Cell Biol. 1989 Apr;108(4):1245–1256. doi: 10.1083/jcb.108.4.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Beckers C. J., Block M. R., Glick B. S., Rothman J. E., Balch W. E. Vesicular transport between the endoplasmic reticulum and the Golgi stack requires the NEM-sensitive fusion protein. Nature. 1989 Jun 1;339(6223):397–398. doi: 10.1038/339397a0. [DOI] [PubMed] [Google Scholar]
  11. Beckers C. J., Keller D. S., Balch W. E. Semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex. Cell. 1987 Aug 14;50(4):523–534. doi: 10.1016/0092-8674(87)90025-0. [DOI] [PubMed] [Google Scholar]
  12. Bourne H. R. Do GTPases direct membrane traffic in secretion? Cell. 1988 Jun 3;53(5):669–671. doi: 10.1016/0092-8674(88)90081-5. [DOI] [PubMed] [Google Scholar]
  13. Braell W. A. Fusion between endocytic vesicles in a cell-free system. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1137–1141. doi: 10.1073/pnas.84.5.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Burgoyne R. D. Small GTP-binding proteins. Trends Biochem Sci. 1989 Oct;14(10):394–396. doi: 10.1016/0968-0004(89)90281-8. [DOI] [PubMed] [Google Scholar]
  15. Calés C., Hancock J. F., Marshall C. J., Hall A. The cytoplasmic protein GAP is implicated as the target for regulation by the ras gene product. Nature. 1988 Apr 7;332(6164):548–551. doi: 10.1038/332548a0. [DOI] [PubMed] [Google Scholar]
  16. Casey P. J., Solski P. A., Der C. J., Buss J. E. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8323–8327. doi: 10.1073/pnas.86.21.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. DeFeo-Jones D., Scolnick E. M., Koller R., Dhar R. ras-Related gene sequences identified and isolated from Saccharomyces cerevisiae. Nature. 1983 Dec 15;306(5944):707–709. doi: 10.1038/306707a0. [DOI] [PubMed] [Google Scholar]
  18. Deretic D., Hamm H. E. Topographic analysis of antigenic determinants recognized by monoclonal antibodies to the photoreceptor guanyl nucleotide-binding protein, transducin. J Biol Chem. 1987 Aug 5;262(22):10839–10847. [PubMed] [Google Scholar]
  19. Fischer von Mollard G., Mignery G. A., Baumert M., Perin M. S., Hanson T. J., Burger P. M., Jahn R., Südhof T. C. rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1988–1992. doi: 10.1073/pnas.87.5.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gibbs J. B., Marshall M. S. The ras oncogene--an important regulatory element in lower eucaryotic organisms. Microbiol Rev. 1989 Jun;53(2):171–185. doi: 10.1128/mr.53.2.171-185.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gibbs J. B., Schaber M. D., Allard W. J., Sigal I. S., Scolnick E. M. Purification of ras GTPase activating protein from bovine brain. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5026–5030. doi: 10.1073/pnas.85.14.5026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Hamm H. E., Deretic D., Arendt A., Hargrave P. A., Koenig B., Hofmann K. P. Site of G protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit. Science. 1988 Aug 12;241(4867):832–835. doi: 10.1126/science.3136547. [DOI] [PubMed] [Google Scholar]
  24. Hamm H. E., Deretic D., Hofmann K. P., Schleicher A., Kohl B. Mechanism of action of monoclonal antibodies that block the light activation of the guanyl nucleotide-binding protein, transducin. J Biol Chem. 1987 Aug 5;262(22):10831–10838. [PubMed] [Google Scholar]
  25. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  26. 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. doi: 10.1002/j.1460-2075.1987.tb02750.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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. doi: 10.1002/j.1460-2075.1989.tb03524.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kataoka T., Powers S., Cameron S., Fasano O., Goldfarb M., Broach J., Wigler M. Functional homology of mammalian and yeast RAS genes. Cell. 1985 Jan;40(1):19–26. doi: 10.1016/0092-8674(85)90304-6. [DOI] [PubMed] [Google Scholar]
  29. Kitayama H., Sugimoto Y., Matsuzaki T., Ikawa Y., Noda M. A ras-related gene with transformation suppressor activity. Cell. 1989 Jan 13;56(1):77–84. doi: 10.1016/0092-8674(89)90985-9. [DOI] [PubMed] [Google Scholar]
  30. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  31. Lowy D. R., Willumsen B. M. Protein modification: new clue to Ras lipid glue. Nature. 1989 Oct 5;341(6241):384–385. doi: 10.1038/341384a0. [DOI] [PubMed] [Google Scholar]
  32. Marshall M. S., Hill W. S., Ng A. S., Vogel U. S., Schaber M. D., Scolnick E. M., Dixon R. A., Sigal I. S., Gibbs J. B. A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity. EMBO J. 1989 Apr;8(4):1105–1110. doi: 10.1002/j.1460-2075.1989.tb03480.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Melançon P., Glick B. S., Malhotra V., Weidman P. J., Serafini T., Gleason M. L., Orci L., Rothman J. E. Involvement of GTP-binding "G" proteins in transport through the Golgi stack. Cell. 1987 Dec 24;51(6):1053–1062. doi: 10.1016/0092-8674(87)90591-5. [DOI] [PubMed] [Google Scholar]
  34. Michaeli T., Field J., Ballester R., O'Neill K., Wigler M. Mutants of H-ras that interfere with RAS effector function in Saccharomyces cerevisiae. EMBO J. 1989 Oct;8(10):3039–3044. doi: 10.1002/j.1460-2075.1989.tb08454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Milburn M. V., Tong L., deVos A. M., Brünger A., Yamaizumi Z., Nishimura S., Kim S. H. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins. Science. 1990 Feb 23;247(4945):939–945. doi: 10.1126/science.2406906. [DOI] [PubMed] [Google Scholar]
  36. Molenaar C. M., 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. doi: 10.1002/j.1460-2075.1988.tb02903.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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. doi: 10.1083/jcb.107.3.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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. doi: 10.1083/jcb.109.6.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pai E. F., Kabsch W., Krengel U., Holmes K. C., 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. doi: 10.1038/341209a0. [DOI] [PubMed] [Google Scholar]
  40. Rivier J., McClintock R., Galyean R., Anderson H. Reversed-phase high-performance liquid chromatography: preparative purification of synthetic peptides. J Chromatogr. 1984 Apr 24;288(2):303–328. doi: 10.1016/s0021-9673(01)93709-4. [DOI] [PubMed] [Google Scholar]
  41. Ruohola H., Kabcenell A. K., Ferro-Novick S. Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant. J Cell Biol. 1988 Oct;107(4):1465–1476. doi: 10.1083/jcb.107.4.1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sakakibara S., Shimonishi Y., Kishida Y., Okada M., Sugihara H. Use of anhydrous hydrogen fluoride in peptide synthesis. I. Behavior of various protective groups in anhydrous hydrogen fluoride. Bull Chem Soc Jpn. 1967 Sep;40(9):2164–2167. doi: 10.1246/bcsj.40.2164. [DOI] [PubMed] [Google Scholar]
  43. Salminen A., Novick P. J. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. doi: 10.1016/0092-8674(87)90455-7. [DOI] [PubMed] [Google Scholar]
  44. Salminen A., Novick P. J. 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. doi: 10.1083/jcb.109.3.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Santos E., Nebreda A. R. Structural and functional properties of ras proteins. FASEB J. 1989 Aug;3(10):2151–2163. doi: 10.1096/fasebj.3.10.2666231. [DOI] [PubMed] [Google Scholar]
  46. Schaber M. D., Garsky V. M., Boylan D., Hill W. S., Scolnick E. M., Marshall M. S., Sigal I. S., Gibbs J. B. Ras interaction with the GTPase-activating protein (GAP). Proteins. 1989;6(3):306–315. doi: 10.1002/prot.340060313. [DOI] [PubMed] [Google Scholar]
  47. Schafer W. R., Kim R., Sterne R., Thorner J., Kim S. H., Rine J. Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science. 1989 Jul 28;245(4916):379–385. doi: 10.1126/science.2569235. [DOI] [PubMed] [Google Scholar]
  48. Schmitt H. D., 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. doi: 10.1016/0092-8674(88)90579-x. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. 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]
  51. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Scolnick E. M. Identification of effector residues and a neutralizing epitope of Ha-ras-encoded p21. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4725–4729. doi: 10.1073/pnas.83.13.4725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stone J. C., Vass W. C., Willumsen B. M., Lowy D. R. p21-ras effector domain mutants constructed by "cassette" mutagenesis. Mol Cell Biol. 1988 Aug;8(8):3565–3569. doi: 10.1128/mcb.8.8.3565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tanaka K., Nakafuku M., Satoh T., Marshall M. S., Gibbs J. B., Matsumoto K., Kaziro Y., Toh-e A. S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein. Cell. 1990 Mar 9;60(5):803–807. doi: 10.1016/0092-8674(90)90094-u. [DOI] [PubMed] [Google Scholar]
  54. Tong L. A., de Vos A. M., Milburn M. V., Jancarik J., Noguchi S., Nishimura S., Miura K., Ohtsuka E., Kim S. H. Structural differences between a ras oncogene protein and the normal protein. Nature. 1989 Jan 5;337(6202):90–93. doi: 10.1038/337090a0. [DOI] [PubMed] [Google Scholar]
  55. 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. doi: 10.1073/pnas.84.23.8210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  57. Trahey M., Wong G., Halenbeck R., Rubinfeld B., Martin G. A., Ladner M., Long C. M., Crosier W. J., Watt K., Koths K. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. doi: 10.1126/science.3201259. [DOI] [PubMed] [Google Scholar]
  58. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  59. Wagner P., Molenaar C. M., Rauh A. J., Brökel R., Schmitt H. D., Gallwitz D. Biochemical properties of the ras-related YPT protein in yeast: a mutational analysis. EMBO J. 1987 Aug;6(8):2373–2379. doi: 10.1002/j.1460-2075.1987.tb02514.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Walworth N. C., Goud B., Kabcenell A. K., Novick P. J. Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic. EMBO J. 1989 Jun;8(6):1685–1693. doi: 10.1002/j.1460-2075.1989.tb03560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Willumsen B. M., Papageorge A. G., Kung H. F., Bekesi E., Robins T., Johnsen M., Vass W. C., Lowy D. R. Mutational analysis of a ras catalytic domain. Mol Cell Biol. 1986 Jul;6(7):2646–2654. doi: 10.1128/mcb.6.7.2646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wilson D. W., Wilcox C. A., Flynn G. C., Chen E., Kuang W. J., Henzel W. J., Block M. R., Ullrich A., Rothman J. E. A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast. Nature. 1989 Jun 1;339(6223):355–359. doi: 10.1038/339355a0. [DOI] [PubMed] [Google Scholar]
  63. Zahraoui A., Touchot N., Chardin P., Tavitian A. The human Rab genes encode a family of GTP-binding proteins related to yeast YPT1 and SEC4 products involved in secretion. J Biol Chem. 1989 Jul 25;264(21):12394–12401. [PubMed] [Google Scholar]
  64. de Vos A. M., Tong L., Milburn M. V., Matias P. M., Jancarik J., Noguchi S., Nishimura S., Miura K., Ohtsuka E., Kim S. H. Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. Science. 1988 Feb 19;239(4842):888–893. doi: 10.1126/science.2448879. [DOI] [PubMed] [Google Scholar]

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