Subcellular distribution and membrane association of rho-related
small gtp-binding proteins in the kidney cortex.
Boivin, Dominique, and Richard B[acute]eliveau.
Laboratoire de Membranologie, Groupe de Recherche en Transport
Membranaire, D[acute]epartement de Chimie-Biochimie,
Universit[acute]e du Qu[acute]ebec [grave]a Montr[acute]eal, P.O. Box
8888, Station A, Montr[acute]eal, Qu[acute]ebec, Canada H3C 3P8
APStracts 2:0042F, 1995.
We have examined the subcellular distribution of Rho-related small
GTP-binding proteins in the kidney. RhoA, CDC42, and Rac1 small GTP
-binding proteins were found to be expressed at high levels in rat
outer kidney cortex. Western blot analysis showed that these proteins
were predominantly 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 small fraction was associated with cytoskeletal
elements. A GDP-dissociation inhibitor specific for the Rho family
(RhoGDI) was also identified and found to be located exclusively in
the cytosol. Upon fractionation of kidney cytosol on anion-exchange
chromatography, RhoA and CDC42 proteins eluted in two major well
-resolved peaks that coeluted with the RhoGDI protein, suggesting that
they form heterodimers. Association of RhoA and CDC42 with RhoGDI was
further suggested by coelution of these proteins with RhoGDI at an
estimated size of about 45 kDa upon gel filtration chromatography.
However, a second peak of RhoA eluted as a 20-kDa protein, indicating
that not all RhoA is complexed to RhoGDI. Addition of RhoA- and
CDC42-enriched fractions to purified membranes from kidney cortex
resulted in their translocation to the membranes and their carboxyl
methylation. Both processes were stimulated by GTP[gamma]S.
Methylation inhibitors had no effect on the translocation of RhoA to
membranes, suggesting that this covalent modification is not required
for association to the membrane. Our 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 plasma membranes.
Received 26 September 1994; accepted in final form 21 February
1995.
APS Manuscript Number F342-4.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 4 April 1995.