Characterization of the association of the actin-binding protein, IQGAP, and activated Cdc42 with Golgi membranes.
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.
McCallum
S J
SJ
Department of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
Erickson
J W
JW
Cerione
R A
RA
D000818:Animals; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000378:metabolism*; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D020764:cdc42 GTP-Binding Protein
D018797:Cell Cycle Proteins; D020690:GTPase-Activating Proteins; D011506:Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein