Molecular Dissection of Guanine Nucleotide Dissociation Inhibitor Function in Vivo
Rab-INDEPENDENT BINDING TO MEMBRANES AND ROLE OF RAB RECYCLING FACTORS*
- From the ‡Departments of Cell and Molecular Biology-IMM 11, The Scripps Research Institute, La Jolla, California 92037 and the ¶Division of Cellular and Molecular Medicine, The Howard Hughes Medical Institute, University of California, San Diego, School of Medicine, La Jolla, California 92093-0668
Abstract
Guanine nucleotide dissociation inhibitor (GDI) is an essential protein required for the recycling of Rab GTPases mediating the targeting and fusion of vesicles in the exocytic and endocytic pathways. Using site-directed mutagenesis of yeastGDI1, we demonstrate that amino acid residues required for Rab recognition in vitro are critical for function in vivo in Saccharomyces cerevisiae. Analysis of the effects of Rab-binding mutants on function in vivo reveals that only a small pool of recycling Rab protein is essential for growth, and that the rates of recycling of distinct Rabs are differentially sensitive to GDI. Furthermore, we find that membrane association of Gdi1p is Rab-independent. Mutant Gdi1 proteins unable to bind Rabs were able to associate with cellular membranes as efficiently as wild-type Gdi1p, yet caused a striking loss of the endogenous cytosolic Gdi1p-Rab pools leading to dominant inhibition of growth when expressed at levels of the normal, endogenous pool. These results demonstrate a potential role for a new recycling factor in the retrieval of Rab-GDP from membranes, and illustrate the importance of multiple effectors in regulating GDI function in Rab delivery and retrieval from membranes.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants GM33301 (to W. E. B.) and Core B in CA 58689 (to S. D. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed. E-mail:webalch{at}scripps.edu.
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↵‖ Investigator of the Howard Hughes Medical Institute.
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↵** Supported as a Postdoctoral Fellow of the American Cancer Society.
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↵2 P. Luan, W. E. Balch, S. D. Emr, and C. G. Burd, unpublished observations.
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↵3 P. Luan, W. E. Balch, S. D. Emr, and C. G. Burd, unpublished results.
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↵4 W. E. Balch, unpublished data.
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↵5 W. E. Balch, unpublished observations.
- Abbreviations:
- GDI
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guanine nucleotide dissociation inhibitor
- SCR
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sequence conserved region
- ER
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endoplasmic reticulum
- HA
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hemagglutinin
- CPY
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carboxypeptidase Y
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- Received November 10, 1998.
- Revision received March 10, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
