Plant Molecular Biology

, Volume 33, Issue 6, pp 1025–1035 | Cite as

The presence of a Sar1 gene family in Brassica campestris that suppresses a yeast vesicular transport mutation Sec12-1

  • Woe Yeon Kim
  • Na Eun Cheong
  • Dae Yeop Je
  • Min Gab Kim
  • Choe Oh Lim
  • Jeong Dong Bahk
  • Moo Je Cho
  • Sang Yeol Lee*


Two new members (Bsar1a and Bsar1b) of the Sar1 gene family have been identified from a flower bud cDNA library of Brassica campestris and their functional characteristics were analyzed. The two clones differ from each other at 14 positions of the 193 amino acid residues deduced from their coding region. The amino acid sequences of Bsar1a and Bsar1b are most closely related to the Sar1 family, genes that function early in the process of vesicle budding from the endoplasmic reticulum (ER). The sequences contain all the conserved motifs of the Ras superfamily (G1–G4 motifs) as well as the distinctive structural feature near the C-terminus that is Sar1 specific. Our phylogenetic analysis confirmed that these two clones can indeed be considered members of the Sar1 family and that they have a close relationship to the ARF family. The Bsar1 proteins, expressed in Escherichia coli, cross-reacted with a polyclonal antibody prepared against Saccharomyces cerevisiae Sar1 protein. It also exhibited GTP-binding activity. Genomic Southern blot analysis, using the 3'-gene-specific regions of the Bsar1 cDNAs as probes, revealed that the two cDNA clones are members of a B. campestris Sar1 family that consists of 2 to 3 genes. RNA blot analysis, using the same gene-specific probes, showed that both genes are expressed with similar patterns in most tissues of the plant, including leaf, stem, root, and flower buds. Furthermore, when we placed the two Bsar1 genes under the control of the yeast pGK1 promoter into the temperature-sensitive mutant yeast strain S. cerevisiae Sec12-1, they suppressed the mutation which consists of a defect in vesicle transport. The amino acid sequence similarity, the GTP-binding activity, and the functional suppression of the yeast mutation suggest that the Bsar1 proteins are functional homologues of the Sar1 protein in S. cerevisiae and that they may perform similar biological functions.

Brassica Sar1-like cDNAs small GTP-binding protein suppression yeast Sec12-1 mutant 


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  1. 1.
    Anai T, Hasegawa K, Watanabe Y, Uchimiya H, Ishizaki R, Matusui M: Isolation and analysis of cDNAs encoding small GTP-binding proteins ofArabidopsis thaliana. Gene 108: 259–264 (1991).Google Scholar
  2. 2.
    Barlowe C, d'Enfert C, Schekman R: Purification and characterization of Sar1p, a small GTP binding protein required for transport vesicle formation from the endoplasmic reticulum. J Biol Chem 268: 873–879 (1993).Google Scholar
  3. 3.
    Bourne HR, Sanders DA, McCormick F: The GTPase superfamily: conserved structure and molecular mechanism. Nature 349: 117–127 (1991).Google Scholar
  4. 4.
    Capon DJ, Chen EY, Levinson AD, Seeburg PH, Goeddel DV: Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature 302: 33–37 (1983).Google Scholar
  5. 5.
    Chardin P, Madaule P, Tavitian A: Coding sequence of human rho cDNAs clone 6 and clone 9. Nucl Acids Res 16: 2717 (1988).Google Scholar
  6. 6.
    Chavrier P, Parton RG, Hauri HP, Simons K, Zerial M: Localization of low molecular weight GTP-binding proteins to exocytic and endocytic compartments. Cell 62: 317–329 (1990).Google Scholar
  7. 7.
    Dascher C, Ossig R, Gallwitz D, Schmitt HD: Identification and structure of four yeast genes (SLY) that able to suppress the functional loss of Ypt1, a member of the RAS superfamily. Mol Cell Biol 11: 872–885 (1991).Google Scholar
  8. 8.
    Davies C: Cloning and characterization of a tomato GTPaselike gene related to yeast and Arabidopsis genes involved in vesicular transport. Plant Mol Biol 24: 525–531 (1994).Google Scholar
  9. 9.
    Dellaporta SL, Wood J, Hicks JB: A plant DNA minipreparation. Version II. Plant Mol Biol Rep 1: 19–22 (1983).Google Scholar
  10. 10.
    Donaldson JG, Cassel D, Kahn RA, Klausner RD: ADPribosylation factor, a small GTP-binding protein, is required for binding coatomer proteinβ-cop to Golgi membranes. Proc Natl Acad Sci USA 89: 6408–6412 (1992).Google Scholar
  11. 11.
    d'Enfert C, Gensse M, Gaillardin C: Fission yeast and a plant have functional homologues of the Sar 1 and Sec12 proteins involved in ER to Golgi traffic in budding yeast. EMBO J 11: 4205–4211 (1992).Google Scholar
  12. 12.
    d'Enfert C, Wuestehube LJ, Lila T, Schekman R: Sec12p dependent membrane binding of the small GRP-binding protein Sar 1p promotes formation of transport vesicles from the ER. J Cell Biol 114: 663–670 (1991).Google Scholar
  13. 13.
    Fukui Y, Kaziro Y: Molecular cloning and sequence analysis of a ras gene from Schizosaccharomyces pombe. EMBO J 4: 687–691 (1985).Google Scholar
  14. 14.
    Gallwitz D, Donath C, Sander C: A yeast gene containing a protein homologous to the human c-has/bas proto-oncogene product. Nature 306: 704–707 (1983).Google Scholar
  15. 15.
    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 8: 1427–1432 (1989).Google Scholar
  16. 16.
    Hicke L, Yoshihisa T, Schekman R: Sec23p and a novel 105 kD protein function as a multimeric complex to promote vesicular budding and protein transport from the ER. Mol Biol Cell 3: 667–676 (1992).Google Scholar
  17. 17.
    Ito H, Fulude Y, Murata K, Kimura A: Transformation of intact yeast cells treated with alkali cations. J Bact 153: 163–169 (1983).Google Scholar
  18. 18.
    Kaiser C: Protein transport from the endoplasmic reticulum to the Golgi apparatus. In: Loh YP (ed) Mechanisms of Intracellular Trafficking and Processing of Proproteins, pp. 79–101. CRC Press, Boca Raton, FL. (1993).Google Scholar
  19. 19.
    Kahn RA, Kern FG, Clark J, Gelmann EP, Rulka C: Human ADP-ribosylation factors. A functionally conserved family of GTP-binding proteins. J Biol Chem 266: 2604–2614 (1991).Google Scholar
  20. 20.
    Kim WY, Cheong NE, Lee DC, Lee KO, Je DY, Bahk JD, Cho MJ, Lee SY: Isolation of an additional soybean cDNA encoding a Ypt/rab-related small GTP-binding protein and its functional comparison to Sypt using a yeast ypt 1-1 mutant. Plant Mol Biol 31: 783–792 (1996).Google Scholar
  21. 21.
    Kuge O, Dascher C, Orci L, Rowe T, Amherdt M, Plunter H, Avazzola M, Tanigawa JE, Balch WE: Sar 1 promotes vesicle budding from the endoplasmic reticulum but not Golgi compartments. J Cell Biol 125: 51–65 (1994).Google Scholar
  22. 22.
    Lim CO, Kim MG, Hwang I, Cho MJ: Expressed sequence tags of a Chinese cabbage (Brassica campestris L.ssp. pekinensis) flower bud cDNA library. Plant Physiol 111: 577–588 (1996).Google Scholar
  23. 23.
    Madaule P, Axel R, Myers AM: Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci USA 84: 779–783 (1987).Google Scholar
  24. 24.
    Molenaar CMT, 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. 7: 971–976 (1988).Google Scholar
  25. 25.
    Nagano Y, Murai N, Matsuno R, Sasaki Y: Isolation and characterization of cDNAs that encode eleven small GTP binding proteins from Pisum sativum. Plant Cell Physiol 34: 447–455 (1993).Google Scholar
  26. 26.
    Nakano A, Muramatsu M: A novel GTP-binding protein, Sar 1p, is involved in transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Biol 109: 2677–2691 (1989).Google Scholar
  27. 27.
    Nakano A, Brada D, Schekman R: A membrane glycoprotein, Sec12p, is required for protein transport from the ER to the Golgi apparatus in yeast. J Cell Biol 107: 851–863 (1988).Google Scholar
  28. 28.
    Nuoffer C, Balch WE: GTPase; Multifunctional molecular switches regulating vesicular traffic. Annu Rev Biochem 63: 949–990 (1994).Google Scholar
  29. 29.
    Oka T, Nakano A: Inhibition of GTP hydrolysis by Sar 1p causes accumulation of vesicles that are a functional interemetiate of the ER-to-Golgi transport in yeast. J Cell Biol 124: 425–434 (1994).Google Scholar
  30. 30.
    Oka T, Nishikawa S, Nakano A: Reconstitution of GTPbinding Sar 1 protein function in ER to Golgi transport. J Cell Biol 114: 671–679 (1991).Google Scholar
  31. 31.
    Powers S, Kataoka T, Fasano O, Goldfarb m, Strathern J, Broach J, Wigler M: Genes in Saccharomyces cerevisiae encoding proteins with domains homologous to the mammalian ras proteins. Cell 36: 607–612 (1984).Google Scholar
  32. 32.
    Pryer NK, Wuestehube LJ, Schekman R: Vesicle-mediated protein sorting. Annu Rev Biochem 61: 471–516 (1992).Google Scholar
  33. 33.
    Rose MD, Broach JR: Cloning genes by complementation in yeast. Meth Enzymol 194: 195–230 (1991).Google Scholar
  34. 34.
    Salama NR, Yeung T, Schekman R: The Sec13p complex and reconstitution of vesicule budding from the ER with purified cytosolic proteins. EMBO J 12: 4073–4082 (1993).Google Scholar
  35. 35.
    Schmitt HD, Wager 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 47: 401–412 (1986).Google Scholar
  36. 36.
    Segev N, Mulholland J, Botstein D: The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell 52: 915–924 (1988).Google Scholar
  37. 37.
    Sewell JL, Kahn RA: Sequences of the bovine and yeast ADPribosylation factor and comparison to other GTP binding proteins. Proc Natl Acad Sci USA 85: 4620–4624 (1988).Google Scholar
  38. 38.
    Shen KA, Hammond CM, Moore HH: Molecular analysis of Sar1 related cDNAs from a mouse pituitary cell line. FEBS Lett 335: 380–385 (1993).Google Scholar
  39. 39.
    Stearns T, Kahn RA, Botstein D, Hoyt MA: ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes. Mol Cell Biol 10: 6690–6699 (1990).Google Scholar
  40. 40.
    Terryn N, Van Montagu MV, Inzé D: GTP-binding proteins in plants. Plant Mol Biol 22: 143–152 (1993).Google Scholar
  41. 41.
    Yoshihisa T, Barlowe C, Schekman R: Recruitment of a GTPase activating protein in vesicle budding from the endoplasmic reticulum. Science 259: 1466–1468 (1993).Google Scholar
  42. 42.
    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 264: 12394–12401 (1989).Google Scholar
  43. 43.
    Zerial M, Stenmark H: Rab GTPases in vesicular transport. Curr Biol 5: 613–620 (1993).Google Scholar

Copyright information

© Kluwer Academic Publishers 1997

Authors and Affiliations

  • Woe Yeon Kim
    • 1
  • Na Eun Cheong
    • 1
  • Dae Yeop Je
    • 1
  • Min Gab Kim
    • 1
  • Choe Oh Lim
    • 1
  • Jeong Dong Bahk
    • 1
  • Moo Je Cho
    • 1
  • Sang Yeol Lee*
    • 1
  1. 1.Department of Biochemistry, Plant Molecular Biology and Biotechnology Research CenterGyeongsang National UniversityChinjuKorea

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