Characterization of the Association of the Actin-binding Protein, IQGAP, and Activated Cdc42 with Golgi Membranes*
- From the Departments of ‡Biochemistry, Molecular and Cell Biology and §Molecular Medicine, Cornell University, Ithaca, New York 14853
Abstract
IQGAP is a recently identified actin-binding protein, which is a putative target for the Cdc42 and Rac GTP-binding proteins. Cdc42 was localized to the Golgi (Erickson, J. W., Zhang, C., Kahn, R. A., Evans, T., and Cerione, R. A. (1996)J. Biol. Chem. 271, 26850–26854), and here we show by immunofluorescence that IQGAP has a perinuclear localization, that it can be co-immunoprecipitated with Cdc42 from Golgi-enriched fractions, and that purified Golgi membranes are recognized by specific antibodies raised against IQGAP and Cdc42 in negative-stain immunogold electron microscopy experiments. Addition of activated, recombinant Cdc42 or solubilization of endogenous Cdc42 from Golgi membranes by the Rho-GDP dissociation inhibitor protein fails to solubilize IQGAP, suggesting that it associates with these membranes in a Cdc42-independent manner. Detergent solubilization of Golgi membranes leaves IQGAP and actin in an insoluble pellet but releases Cdc42 to the supernatant, whereas treatments that release actin from this detergent-insoluble pellet also release IQGAP. Addition of the COOH-terminal half of the IQGAP protein, which contains the Cdc42-binding domain, removes Cdc42 from Golgi membranes in a dose-dependent manner. These data suggest that IQGAP and Cdc42 are part of a cytoskeletal complex in Golgi membranes that may mediate Cdc42-regulated effects on the actin cytoskeleton in these membranes.
Footnotes
-
↵* 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.
-
↵¶ To whom correspondence should be addressed: Dept. of Molecular Medicine, Veterinary Medical Center, Cornell University, Ithaca, NY 14853-6401. Tel.: 607-253-3888; Fax: 607-253-3659.
-
↵2 T. Nomanbhoy and R. A. Cerione, manuscript in preparation.
-
↵3 M. Osman and R. A. Cerione, J. Cell Biol., in press.
-
↵4 S. J. McCallum, J. W. Erickson, and R. A. Cerione, unpublished data.
- Abbreviations:
- RhoGDI
-
Rho GDP-dissociation inhibitor
- GRD
-
RasGAP-related domain
- CHO
-
Chinese hamster ovary
- GST
-
glutathione S-transferase
- PI 3-K
-
phosphatidylinositol 3-kinase
- DTT
-
dithiothreitol
- PLD
-
phospholipase D
- HA
-
hemagglutinin
- PAGE
-
polyacrylamide gel electrophoresis
- GTPγS
-
guanosine 5′-3-O-(thio)triphosphate
- CHAPS
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
-
- Received January 22, 1998.
- Revision received June 4, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
