Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding

Abstract

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.

Figure 1

Localization of GFP-tagged Ras and Rho proteins in MDCK cells. (a–i) MDCK cells were transiently transfected with GFP-tagged constructs of the indicated GTPase and imaged alive 24 h later by digital epifluorescence microscopy using a cooled CCD camera. (j) Listed are the hypervariable regions of each GTPase with the CAAX motif underlined, potential palmitoylation sites shown in outline, acidic residues (gray), and basic residues (bold). Bars, 10 μm.

Figure 2

(a–i) Localization of GFP-tagged Ras and Rho proteins in COS-1 cells. COS-1 cells were transfected with the indicated GFP fusion protein and imaged as in Fig. 1. The insets (a–c) show an enlargement of the ER. (h) Cells transfected with GFP-RhoB were treated with BODIPY TR-ceremide for 30 min before imaging, such that the Golgi appears red in nontransfected cells, and colocalization with GFP-RhoB could be scored as yellow. (j) Cells transfected with GFP-TC10 were treated with Texas red–conjugated transferrin for 30 min before imaging, revealing colocalization in endosomes. Motile peri-Golgi vesicles carrying GFP-RhoB (g) and endosomes carrying GFP-TC10 (i) were best compared by time-lapse imaging as QuickTime^® movies available at http://www.jcb.org/cgi/content/full/152/1/111/DC1). Compare also the QuickTime^® movies of GFP-H-Ras and GFP-pCdc42hs. Bars, 10 μm.

Figure 3

The hypervariable regions of Rho GTPases are sufficient for differential membrane targeting which is independent of the length of the prenyl chain. MDCK cells were transiently transfected with the indicated constructs that included GFP extended at the COOH terminus with the hypervariable regions (last 20 amino acids) of pCdc42hs (a), bCdc42hs (b), Rac1 (c), and RhoA (d) or amino acids 73–193 of RhoA (e). (f) A doubly chimeric fusion protein consisting of GFP followed by amino acids 1–188 of TC10 and then amino acids 172–191 of pCdc42hs. (g and h) The last amino acid of the hypervariable regions of pCdc42hs (g) and Rac1 (h) were changed from L to M to switch modification from geranylgeranylation to farnesylation. Bars, 10 μm.

Figure 4

Localization of K-Ras4B and Rho GTPases in stably transfected cells. Stable transformants of ECV304 or PAE cells expressing the indicated GFP-tagged fusion proteins were selected with G418 and cytofluorometry and imaged alive by digital epifluorescence microscopy using a cooled CCD camera. Bars, 10 μm.

Figure 5

Localization of endogenous Rho GTPases and RhoGDI. (a) Indirect immunofluorescence: untransfected MDCK cells were grown on coverslips, fixed with 3% paraformaldehyde (i–iv, and vi) or ice-cold methanol/acetone (1:1; v), permeabilized with Triton X-100 (i, ii, and iv) or saponin (iii and vi), and stained for the indicated protein as described in Materials and Methods. (b) Subcellular fractionation: COS-1 cell cytosolic and membrane fractions were analyzed by immunoblot for the indicated proteins, and relative amounts were quantitated by a PhosphorImager. Results are expressed as the percentage of the endogenous protein detected in the membrane fraction and are given as mean ± SEM (n = 8). Bars, 10 μm.

Figure 6

Translocation of Rho GTPases to lamellipodia. ECV304 cells stably expressing GFP-tagged Rac1 (a and d), pCdc42hs (b and e), or RhoA (c and f) were transiently transfected with oncogenic Dbl (d–f) or vector alone (a–c) and imaged alive after 24 h as in the legend to Fig. 4. Bars, 10 μm.

Figure 7

Relative expression levels of Rho GTPases and RhoGDIα affect the localization of some, but not all, Rho proteins. MDCK cells were transiently transfected with 0.2 μg (a i–h i) or 5 μg (a ii–h iii) of plasmid DNA encoding the indicated GFP-tagged GTPase along with 10 μg of vector (pcDNA3.1) DNA (a ii–h ii) or pcDNA3.1-RhoGDIα DNA (a iii–h iii). 24 h after transfection, the cells were imaged alive as in the legend to Fig. 1. Bars, 10 μm.

Figure 8

The role of palmitoylation on localization and RhoGDIα binding of Rho GTPases. COS-1 (a–e, i, and j) or MDCK (f–h) cells were transiently transfected with the indicated GFP-tagged GTPase in the absence (a, c, e, f, h, and i) or presence (b, d, g, and j) of 25 μM 2BP and imaged alive after 24 h as described in the legend to Fig. 1. (f–h) GFP-tagged RhoA in which the last four amino acids (CLVL) were switched to the last five amino acids of RhoB (CCKVL) was expressed without (f) or with (g) 2BP or coexpressed with excess (1:2 DNA ratio) RhoGDIα (h). Bars, 10 μm.

Figure 9

Dominant negative and some dominant active mutants of Rho proteins do not bind RhoGDIα in vivo. (a) MDCK cells were transiently transfected with the indicated GFP-tagged Rho GTPase mutant without (−GDI) or with (+GDI) cotransfection with RhoGDIα as described in the legend to Fig. 7 and imaged alive after 24 h as described in the legend to Fig. 1. (b) MDCK cells were transiently transfected with the indicated GFP-tagged RhoA allele and imaged alive after 24 h with a Zeiss 510 LSC inverted microscope. Note negatively imaged organelles (i) versus positively imaged organelles (ii–iv). Bars, 10 μm.

Figure 10

Differential binding of Rho GTPases to RhoGDIα. (a, b, and d) COS-1 cells were transiently transfected with the indicated GFP-tagged GTPase without (white bars) or with (black bars) cotransfection with equal amounts of DNA encoding RhoGDIα. After 24 h, each GTPase was immunoprecipitated from cell lysates with an anti-GFP antibody, and immunoprecipitates were immunoblotted (b, inset) for both GFP and RhoGDIα, using ¹²⁵I–protein A and PhosphorImager analysis to quantitate immunoprecipitated proteins. Binding indices are shown in arbitrary units as the ratio of RhoGDIα/GFP-GTPase PhosphorImager volumes and are plotted as the mean ± SEM of n ≥ 4. (a) Wild-type (WT) Rho proteins, (b) pCdc42hs, RhoA, and Rac1 mutation series, (d) chimeric GTPases, and the effect of palmitoylation (introduction of a palmitoylation site into RhoA, RhoA-CCKVL, with or without 2BP, and the removal of palmitoylation sites from RhoB, RhoB189,192S). (c) COS-1 cells were transiently transfected with GFP-tagged Cdc42hs wild-type, Cdc42hs61L, or Cdc42hs12V, and an SRF luciferase reporter construct without (white bars) or with (black bars) coexpression of RhoGDIα. Serum-starved cells were lysed after 16 h, and luciferase activity was measured and normalized to that stimulated by Cdc42hs wild-type in the absence of RhoGDIα. Equivalent expression of Cdc42hs mutants was confirmed by anti-GFP immunoblot. Results shown represent the mean ± SEM, n = 4. The effect of RhoGDIα is significant to P < 0.0001 for Cdc42hs wild-type), P < 0.01 for Cdc42hs12V, and insignificant for Cdc42hs61L.