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Published online 8 January 2001. doi:10.1083/jcb.152.1.111
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© The Rockefeller University Press, 0021-9525/2001//111 $5.00
The Journal of Cell Biology, Volume 152, Number 1, , 2001 111-126

Original Article

Differential Localization of Rho Gtpases in Live Cells

: Regulation by Hypervariable Regions and Rhogdi Binding



David Michaelsona,b, Joseph Sillettia,b, Gretchen Murphyc, Peter D'Eustachioc, Mark Rushc, and Mark R. Philipsa,b

a Department of Medicine, New York University School of Medicine, New York, New York 10016
b Department of Cell Biology, New York University School of Medicine, New York, New York 10016
c Department of Biochemistry, New York University School of Medicine, New York, New York 10016
Departments of Medicine and Cell Biology, MSB251, New York University School of Medicine, 550 First Ave., New York, NY 10016.(212) 263-0759(212) 263-7404

philim01{at}med.nyu.edu

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI){alpha}. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDI{alpha} in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDI{alpha}. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDI{alpha} binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDI{alpha} and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

Key Words: Rho • Rac • Cdc42hs • RhoGDI • green fluorescent protein

© 2001 The Rockefeller University Press

The online version of this article contains supplemental material.

Abbreviations used in this paper: 2BP, 2-bromopalmitate; BFA, brefeldin A; CCD, charge-coupled device; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; GST, glutathione S-transferase; LAMP, lysosome-associated membrane protein; PAE, porcine aortic endothelial; pcCMT, prenylcysteine-directed COOH methyltransferase; PM, plasma membrane; SRF, serum response factor.


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