Abstract
The Arabidopsis Ara proteins belong to the Rab/Ypt family of small GTPases, which are implicated in intracellular vesicular traffic. To understand their specific roles in the cell, it is imperative to identify molecules that regulate the GTPase cycle. Such molecules have been found and characterized in animals and yeasts but not in plants. Using a yeast system, we developed a novel method of functional screening to detect interactions between foreign genes and identified this Rab regulator in plants. We found that the expression of the ARA4 gene in yeast ypt mutants causes exaggeration of the mutant phenotype. By introducing an Arabidopsis cDNA library into the ypt1 mutant, we isolated a clone whose coexpression overcame the deleterious effect of ARA4. This gene encodes an Arabidopsis homolog of the Rab GDP dissociation inhibitor (GDI) and was named AtGDI1. The expression of AtGDI1 complemented the yeast sec19-1 (gdi1) mutation. AtGDI1 is expressed almost ubiquitously in Arabidopsis tissues. The method described here indicates the physiological interaction of two plant molecules, Ara4 and GDI, in yeast and should be applicable to other foreign genes.
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- Anai T., Matsui M., Nomura N., Ishizaki R., Uchimiya H. In vitro mutation analysis of Arabidopsis thaliana small GTP-binding proteins and detection of GAP-like activities in plant cells. FEBS Lett. 1994 Jun 13;346(2-3):175–180. doi: 10.1016/0014-5793(94)80696-9. [DOI] [PubMed] [Google Scholar]
- Araki S., Kikuchi A., Hata Y., Isomura M., Takai Y. Regulation of reversible binding of smg p25A, a ras p21-like GTP-binding protein, to synaptic plasma membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor. J Biol Chem. 1990 Aug 5;265(22):13007–13015. [PubMed] [Google Scholar]
- Bednarek S. Y., Reynolds T. L., Schroeder M., Grabowski R., Hengst L., Gallwitz D., Raikhel N. V. A small GTP-binding protein from Arabidopsis thaliana functionally complements the yeast YPT6 null mutant. Plant Physiol. 1994 Feb;104(2):591–596. doi: 10.1104/pp.104.2.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bucci C., Parton R. G., Mather I. H., Stunnenberg H., Simons K., Hoflack B., Zerial M. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell. 1992 Sep 4;70(5):715–728. doi: 10.1016/0092-8674(92)90306-w. [DOI] [PubMed] [Google Scholar]
- Chuang T. H., Xu X., Knaus U. G., Hart M. J., Bokoch G. M. GDP dissociation inhibitor prevents intrinsic and GTPase activating protein-stimulated GTP hydrolysis by the Rac GTP-binding protein. J Biol Chem. 1993 Jan 15;268(2):775–778. [PubMed] [Google Scholar]
- Dirac-Svejstrup A. B., Soldati T., Shapiro A. D., Pfeffer S. R. Rab-GDI presents functional Rab9 to the intracellular transport machinery and contributes selectivity to Rab9 membrane recruitment. J Biol Chem. 1994 Jun 3;269(22):15427–15430. [PubMed] [Google Scholar]
- Fabry S., Steigerwald R., Bernklau C., Dietmaier W., Schmitt R. Structure-function analysis of small G proteins from Volvox and Chlamydomonas by complementation of Saccharomyces cerevisiae YPT/SEC mutations. Mol Gen Genet. 1995 May 10;247(3):265–274. doi: 10.1007/BF00293194. [DOI] [PubMed] [Google Scholar]
- Ferro-Novick S., Novick P. The role of GTP-binding proteins in transport along the exocytic pathway. Annu Rev Cell Biol. 1993;9:575–599. doi: 10.1146/annurev.cb.09.110193.003043. [DOI] [PubMed] [Google Scholar]
- Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
- Garrett M. D., Kabcenell A. K., Zahner J. E., Kaibuchi K., Sasaki T., Takai Y., Cheney C. M., Novick P. J. Interaction of Sec4 with GDI proteins from bovine brain, Drosophila melanogaster and Saccharomyces cerevisiae. Conservation of GDI membrane dissociation activity. FEBS Lett. 1993 Oct 4;331(3):233–238. doi: 10.1016/0014-5793(93)80343-s. [DOI] [PubMed] [Google Scholar]
- Garrett M. D., Zahner J. E., Cheney C. M., Novick P. J. GDI1 encodes a GDP dissociation inhibitor that plays an essential role in the yeast secretory pathway. EMBO J. 1994 Apr 1;13(7):1718–1728. doi: 10.1002/j.1460-2075.1994.tb06436.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hart M. J., Maru Y., Leonard D., Witte O. N., Evans T., Cerione R. A. A GDP dissociation inhibitor that serves as a GTPase inhibitor for the Ras-like protein CDC42Hs. Science. 1992 Oct 30;258(5083):812–815. doi: 10.1126/science.1439791. [DOI] [PubMed] [Google Scholar]
- Janoueix-Lerosey I., Jollivet F., Camonis J., Marche P. N., Goud B. Two-hybrid system screen with the small GTP-binding protein Rab6. Identification of a novel mouse GDP dissociation inhibitor isoform and two other potential partners of Rab6. J Biol Chem. 1995 Jun 16;270(24):14801–14808. doi: 10.1074/jbc.270.24.14801. [DOI] [PubMed] [Google Scholar]
- Leonard D., Hart M. J., Platko J. V., Eva A., Henzel W., Evans T., Cerione R. A. The identification and characterization of a GDP-dissociation inhibitor (GDI) for the CDC42Hs protein. J Biol Chem. 1992 Nov 15;267(32):22860–22868. [PubMed] [Google Scholar]
- Loraine A. E., Yalovsky S., Fabry S., Gruissem W. Tomato Rab1A homologs as molecular tools for studying Rab geranylgeranyl transferase in plant cells. Plant Physiol. 1996 Apr;110(4):1337–1347. doi: 10.1104/pp.110.4.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Matsui M., Sasamoto S., Kunieda T., Nomura N., Ishizaki R. Cloning of ara, a putative Arabidopsis thaliana gene homologous to the ras-related gene family. Gene. 1989;76(2):313–319. doi: 10.1016/0378-1119(89)90171-6. [DOI] [PubMed] [Google Scholar]
- Matsui Y., Kikuchi A., Araki S., Hata Y., Kondo J., Teranishi Y., Takai Y. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein. Mol Cell Biol. 1990 Aug;10(8):4116–4122. doi: 10.1128/mcb.10.8.4116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moya M., Roberts D., Novick P. DSS4-1 is a dominant suppressor of sec4-8 that encodes a nucleotide exchange protein that aids Sec4p function. Nature. 1993 Feb 4;361(6411):460–463. doi: 10.1038/361460a0. [DOI] [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. doi: 10.1083/jcb.109.6.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Novick P., Field C., Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205–215. doi: 10.1016/0092-8674(80)90128-2. [DOI] [PubMed] [Google Scholar]
- Palmiter R. D. Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undergraded polysomes and messenger ribonucleic acid. Biochemistry. 1974 Aug 13;13(17):3606–3615. doi: 10.1021/bi00714a032. [DOI] [PubMed] [Google Scholar]
- Park Y. S., Song O., Kwak J. M., Hong S. W., Lee H. H., Nam H. G. Functional complementation of a yeast vesicular transport mutation ypt1-1 by a Brassica napus cDNA clone encoding a small GTP-binding protein. Plant Mol Biol. 1994 Dec;26(6):1725–1735. doi: 10.1007/BF00019487. [DOI] [PubMed] [Google Scholar]
- Pfeffer S. R., Dirac-Svejstrup A. B., Soldati T. Rab GDP dissociation inhibitor: putting rab GTPases in the right place. J Biol Chem. 1995 Jul 21;270(29):17057–17059. doi: 10.1074/jbc.270.29.17057. [DOI] [PubMed] [Google Scholar]
- 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]
- Sano H., Seo S., Orudgev E., Youssefian S., Ishizuka K. Expression of the gene for a small GTP binding protein in transgenic tobacco elevates endogenous cytokinin levels, abnormally induces salicylic acid in response to wounding, and increases resistance to tobacco mosaic virus infection. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10556–10560. doi: 10.1073/pnas.91.22.10556. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sasaki T., Kaibuchi K., Kabcenell A. K., Novick P. J., Takai Y. A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein. Mol Cell Biol. 1991 May;11(5):2909–2912. doi: 10.1128/mcb.11.5.2909. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sasaki T., Kato M., Takai Y. Consequences of weak interaction of rho GDI with the GTP-bound forms of rho p21 and rac p21. J Biol Chem. 1993 Nov 15;268(32):23959–23963. [PubMed] [Google Scholar]
- Sasaki T., Kikuchi A., Araki S., Hata Y., Isomura M., Kuroda S., Takai Y. Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein. J Biol Chem. 1990 Feb 5;265(4):2333–2337. [PubMed] [Google Scholar]
- 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]
- 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]
- Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Singer-Krüger B., Stenmark H., Düsterhöft A., Philippsen P., Yoo J. S., Gallwitz D., Zerial M. Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast. J Cell Biol. 1994 Apr;125(2):283–298. doi: 10.1083/jcb.125.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Soldati T., Shapiro A. D., Svejstrup A. B., Pfeffer S. R. Membrane targeting of the small GTPase Rab9 is accompanied by nucleotide exchange. Nature. 1994 May 5;369(6475):76–78. doi: 10.1038/369076a0. [DOI] [PubMed] [Google Scholar]
- Strom M., Vollmer P., Tan T. J., Gallwitz D. A yeast GTPase-activating protein that interacts specifically with a member of the Ypt/Rab family. Nature. 1993 Feb 25;361(6414):736–739. doi: 10.1038/361736a0. [DOI] [PubMed] [Google Scholar]
- Sun G. H., Hirata A., Ohya Y., Anraku Y. Mutations in yeast calmodulin cause defects in spindle pole body functions and nuclear integrity. J Cell Biol. 1992 Dec;119(6):1625–1639. doi: 10.1083/jcb.119.6.1625. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Terryn N., Arias M. B., Engler G., Tiré C., Villarroel R., Van Montagu M., Inzé D. rha1, a gene encoding a small GTP binding protein from Arabidopsis, is expressed primarily in developing guard cells. Plant Cell. 1993 Dec;5(12):1761–1769. doi: 10.1105/tpc.5.12.1761. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Terryn N., Van Montagu M., Inzé D. GTP-binding proteins in plants. Plant Mol Biol. 1993 Apr;22(1):143–152. doi: 10.1007/BF00039002. [DOI] [PubMed] [Google Scholar]
- Tisdale E. J., Bourne J. R., Khosravi-Far R., Der C. J., Balch W. E. GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex. J Cell Biol. 1992 Nov;119(4):749–761. doi: 10.1083/jcb.119.4.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ueda T., Anai T., Tsukaya H., Hirata A., Uchimiya H. Characterization and subcellular localization of a small GTP-binding protein (Ara-4) from Arabidopsis: conditional expression under control of the promoter of the gene for heat-shock protein HSP81-1. Mol Gen Genet. 1996 Mar 20;250(5):533–539. doi: 10.1007/BF02174441. [DOI] [PubMed] [Google Scholar]
- Ullrich O., Horiuchi H., Bucci C., Zerial M. Membrane association of Rab5 mediated by GDP-dissociation inhibitor and accompanied by GDP/GTP exchange. Nature. 1994 Mar 10;368(6467):157–160. doi: 10.1038/368157a0. [DOI] [PubMed] [Google Scholar]
- Ullrich O., Stenmark H., Alexandrov K., Huber L. A., Kaibuchi K., Sasaki T., Takai Y., Zerial M. Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins. J Biol Chem. 1993 Aug 25;268(24):18143–18150. [PubMed] [Google Scholar]
- Verma DPS., Cheon C., 3rd, Hong Z. Small GTP-Binding Proteins and Membrane Biogenesis in Plants. Plant Physiol. 1994 Sep;106(1):1–6. doi: 10.1104/pp.106.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Waldherr M., Ragnini A., Schweyer R. J., Boguski M. S. MRS6--yeast homologue of the choroideraemia gene. Nat Genet. 1993 Mar;3(3):193–194. doi: 10.1038/ng0393-193. [DOI] [PubMed] [Google Scholar]
- Walworth N. C., Brennwald P., Kabcenell A. K., Garrett M., Novick P. Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae. Mol Cell Biol. 1992 May;12(5):2017–2028. doi: 10.1128/mcb.12.5.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Wichmann H., Hengst L., Gallwitz D. Endocytosis in yeast: evidence for the involvement of a small GTP-binding protein (Ypt7p). Cell. 1992 Dec 24;71(7):1131–1142. doi: 10.1016/s0092-8674(05)80062-5. [DOI] [PubMed] [Google Scholar]
- Yoshida K., Nagano Y., Murai N., Sasaki Y. Phytochrome-regulated expression of the genes encoding the small GTP-binding proteins in peas. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6636–6640. doi: 10.1073/pnas.90.14.6636. [DOI] [PMC free article] [PubMed] [Google Scholar]