SUBCELLULAR-DISTRIBUTION AND MEMBRANE ASSOCIATION OF RHO-RELATED SMALL GTP-BINDING PROTEINS IN KIDNEY CORTEX

We have examined the subcellular distribution of Rho-related small GTP -binding proteins in the kidney. RhoA, CDC42, and Rad small GTP-bindin g proteins were found to be expressed at high levels in rat outer kidn ey cortex. Western blot analysis showed that these proteins were predo minantly associated with brush-border and basolateral plasma membranes , with the exception of Rac1 which was localized predominantly in the mitochondria. RhoA and CDC42 were also found in the cytosol, and a sma ll fraction was associated with cytoskeletal elements. A GDP-dissociat ion inhibitor specific for the Rho family (RhoGDI) was also identified and found to be located exclusively in the cytosol. Upon fractionatio n of kidney cytosol with anion-exchange chromatography, RhoA and CDC42 proteins eluted in two major well-resolved peaks that coeluted with t he RhoGDI protein, suggesting that they form heterodimers. Association of RhoA and CDC42 with RhoGDI was further suggested by coelution of t hese proteins with RhoGDI at an estimated size of similar to 45 kDa af ter gelfiltration chromatography. However, a second peak of RhoA elute d as a 20-kDa protein, indicating that not all RhoA is complexed to Rh oGDI. Addition of RhoA- and CDC42-enriched fractions to purified membr anes from kidney cortex resulted in their translocation to the membran es and their carboxyl methylation. Both processes were stimulated by g uanosine 5'-O-(3-thiotriphosphate). Methylation inhibitors had no effe ct on the translocation of RhoA to membranes, suggesting that this cov alent modification is not required for association to the membrane. Ou r results show that in the kidney cortex Rho-related small GTP-binding proteins are preferentially targeted to plasma membranes and suggest that RhoA and CDC42 may cycle between cytoplasmic compartment and plas ma membranes.