Correspondence to: Mark R. Philips, Departments of Medicine and Cell Biology, MSB251, New York University School of Medicine, 550 First Ave., New York, NY 10016. Tel:(212) 263-7404 Fax:(212) 263-0759 E-mail:philim01{at}med.nyu.edu.
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Abstract |
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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). The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDI
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
. 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
binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDI
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
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Introduction |
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Rho proteins are Ras-related GTPases that regulate a variety of cellular processes. More than fifteen mammalian Rho proteins have been described including RhoAE and G, Rac13, two isoforms of Cdc42hs, and TC10. Originally identified as genes homologous to Ras, a great amount of interest in Rho proteins was awakened when
Like all regulatory GTPases, Rho proteins are activated by guanine nucleotide exchange factors (GEFs). A large number of GEFs for Rho proteins have been identified, most of which contain a domain homologous to one found to have GEF activity in the Dbl oncogene (
Like Ras proteins and G protein subunits, Rho GTPases are synthesized as cytosolic proteins but have the capacity to associate with membranes by virtue of a series of posttranslational modifications of a COOH-terminal CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif (
Prenylation of the CAAX motif targets proteins specifically to the endomembrane where they are proteolyzed and methylated (
Examination of the subcellular localization of green fluorescent protein (GFP)-tagged Ras proteins in live cells recently revealed unexpected results that forced a reevaluation of Ras trafficking (
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Materials and Methods |
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Cell Culture and Transfection
COS-1, CHO, NIH3T3, MDCK, and ECV304 cells were obtained from American Type Culture Collection. Porcine aortic endothelial (PAE) cells were obtained from Lena Claesson-Welsh (Chidren's Hospital, Boston, MA). All cells were grown in DME containing 10% FBS (Cellgro) at 5% CO2 and 37°C. Metabolic labeling with [3H]methyl-L-methionine (Amersham Pharmacia Biotech) for methylation assays was performed in methionine-free media, and metabolic labeling with 16-[125I]iodohexadecanoic acid (Marilyn Resh, Memorial Sloane-Kettering Cancer Institute, New York, NY) for palmitoylation was performed with dialyzed FCS. For microscopy, cells were plated, transfected, and imaged in the same 35-mm culture dish that incorporated a no. 1.5 glass coverslipsealed 15-mm cut out on the bottom (MatTek). All transfections were performed 1 d after plating at 50% confluence using SuperFectTM according to the manufacturer's instructions (QIAGEN). Unless otherwise noted, 0.5 µg of DNA was used for each 35-mm dish or 2 µg for each 10-cm dish. In some experiments, brefeldin A (BFA) or 2-bromopalmitate (2BP) (Sigma-Aldrich) was added at the time of transfection. Stably expressing cell lines were established by selecting transfected cells with 0.8 mg/ml G418 (Life Technologies) for 2 wk followed by fluorescence-activated cell sorting for GFP expression. Cells were then G418 selected for another 2 wk. Unless otherwise noted, for coexpression of RhoGDI
, a 1:1 plasmid/DNA ratio was used for coimmunoprecipitation and a 2:1 ratio (RhoGDI
/GTPase) was used for imaging. Control transfections omitting RhoGDI
contained an equivalent amount of vector DNA. Transiently transfected cells were analyzed 1 d after transfection.
Plasmids
cDNAs for the placental isoform of Cdc42hs (pCdc42hs), Rac1, Rac2, RhoA, RhoB, and RhoGDI were obtained from Richard Cerione (Cornell University, Ithaca, NY), Gary Bokoch (Scripps Research Institute, La Jolla, CA), and Alan Hall (University College London, London, UK). TC10 was cloned from human teratocarcinoma cDNA as described previously (
Fluorescence Microscopy
Cells for indirect immunofluorescence were plated into 6-well trays (6 x 35 mm) at a density of 2 x 105 cells per well, containing four coverslips per well. For colocalization studies, the cells were transfected with GFP-tagged Rho proteins the next day using SuperFectTM. Cells on coverslips were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 or 0.5% saponin, or fixed and permeabilized with ice-cold methanol/acetone (1:1, vol/vol) and blocked with 1% milk/0.5% Tween in PBS. For localization of endogenous proteins, the cells were stained with polyclonal antisera to Cdc42hs, RhoA, RhoB, or RhoGDI (Santa Cruz Biotechnology, Inc.) or an anti-Rac1 mAb (Transduction Laboratories). For colocalization, transfected cells were stained with antisera to mannosidase II (Keley Moremen, University of Georgia, Atlanta, GA), or lysosome-associated membrane protein (LAMP)1 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) followed by Texas redconjugated secondary antisera (Jackson ImmunoResearch Laboratories), and mounted on glass slides with Mowiol. For colocalization in live cells, BODIPY TR-ceramide (10 µg/ml) or Texas redconjugated transferrin (100 µg/ml) (Molecular Probes) was incubated with the cell for 30 min before imaging. Live cells were examined 1224 h after transfection with an Axioscope epifluorescence microscope (63x PlanApo 1.4 NA objective; ZEISS) equipped with a Princeton Instruments cooled charge-coupled device (CCD) camera and MetaMorphTM digital imaging software (Universal Imaging Corp.) or a 510 laser scanning confocal microscope (100x PlanApo 1.4 NA objective; ZEISS). Digital images were processed with Adobe Photoshop® v5.0.
Subcellular Fractionation
COS-1 cells were grown to confluence, scraped into 2 ml of hypotonic buffer (10 mM Hepes, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.25 M sucrose, 2 mM PMSF, 10 µg/ml each chymotrypsin, pepstatin, and antipain, 27 µg/ml aprotinin) and disrupted with a ball-bearing homogenizer using 30 passes. Homogenates were cleared of nuclei and unbroken cells (2,500 g, 5 min) and total membrane (P100) and cytosol (S100) were separated by centrifugation (100,000 g, 90 min). Cytosolic and membrane fractions (1 vs. 4 cell equivalents) were analyzed by SDS-PAGE and immunoblotting using polyclonal antisera to RhoGDI (1:500), RhoA (1:200), RhoB (1:200), or Cdc42 (1:200), or an mAb to Rac1 (1:200) followed by rabbit antimouse Ig (1:1,000) and then 125Iprotein A (2.5 x 10-4 mCi/ml). Immunolocalized proteins were visualized and quantitated by a PhosphorImager (Molecular Dynamics).
Coimmunoprecipitation
MDCK cells plated in a 10-cm plate were transiently transfected with pEGFP-C3 containing the desired Rho GTPase insert and either pcDNA3.1-RhoGDI or pcDNA3.1 lacking an insert (1:1 ratio of plasmid DNA). 1 d after transfection, the plates were lysed in 500 µl 1x RIPA (1% Triton X-100, 50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM PMSF, 10 µg/ml each chymotrypsin, pepstatin, and antipain, 27 µg/ml aprotinin). Debris was removed by centrifugation. The supernatant was mixed with an anti-GFP mAb (Boehringer) at 1:500 for 1 h at 4°C. Protein Gagarose beads (20 µl of 1:1 suspension in 1x RIPA; CLONTECH Laboratories, Inc.) was then added for 1 h. Agarose beads were washed two times with 1x RIPA and two times with PBS. Proteins were eluted with 30 µl SDS sample buffer containing 0.5% ß-mercaptoethanol (CLONTECH Laboratories, Inc.) and analyzed by immunoblot for RhoGDI
(2130-kD region; 1:200 anti-RhoGDI
antiserum) and GFP-GTPase (4266-kD region; 1:200 anti-GFP antiserum) using 125Iprotein A (2.5 x 10-4 mCi/ml in phosphotungstic acid) as a secondary reagent. Immunolocalized proteins were visualized and quantitated by a PhosphorImager.
Quantitation of Endogenous Rho GTPases
Recombinant Rac1, Cdc42hs, RhoA, and RhoGDI were produced as glutathione S-transferase (GST) fusion proteins (pGEX2T) in Escherichia coli and cleaved from GST with thrombin. The concentrations of each recombinant protein were determined by densitometric analysis of Coomassie bluestained gels using BSA as a standard. MDCK, COS, and ECV cells were plated on 10-cm plates and grown to confluence. Cells were lysed in 250 µl of SDS sample buffer containing 0.5% ß-mercaptoethanol (CLONTECH Laboratories, Inc.). Various amounts of lysate (210 µl) were analyzed alongside titrated recombinant protein standards by 14% Tris-glycine SDS-PAGE (Novex) and immunoblotted using antisera to Cdc42hs, Rac1, RhoA, or RhoGDI
followed by 125Iprotein A. Immunodetected proteins were quantitated by a PhosphorImager, and cellular concentrations of the various proteins were calculated.
SRF Assay
COS-1 cells were plated in 6-well plates. The next day the cells were transfected using SuperFectTM with 0.2 µg/well SRE-Luciferase plasmid DNA (Promega), 0.4 µg/well of pEGFP DNA containing inserts for wild-type pCdc42hs, pCdc42hs61L, or pCdc42hs12V, and 0.8 µg/well of DNA from either pcDNA3.1 or pcDNA3.1-RhoGDI. After 34 h of recovery, cells were subjected to serum starvation overnight. The next day each well was lysed in 100 µl CCLR detergent. Debris was removed by centrifugation, and 5 µl of each supernatant was mixed with 100 µl Luciferase Assay Reagent (Promega). Luminescence was measured in a luminometer. 20 µl of each supernatant was analyzed by 14% Tris-glycine SDS-PAGE and immunoblotted using antisera to GFP or RhoGDI
and 125Iprotein A, and immunolocalized proteins were quantitated by a PhosphorImager. Luminometer determinations were normalized to GTPase expression.
Online Supplemental Material
Four QuickTime® supplemental videos are available at http://www. jcb.org/cgi/content/full/152/1/111/DC1 showing time-lapse (150x speed) images of GFP-H-Ras, GFP-RhoB, GFP-TC10, and GFP-Cdc42hs expressed in COS-1 cells. Time-lapse digital epifluorescent images were captured from live cells with MetaMorphTM imaging software and converted to avi video files that were edited with Adobe PremiereTM v4.0 and compiled as QuickTime® movies.
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Results |
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Differential Localization of GFP-tagged Rho GTPases
To determine the subcellular location of Rho GTPases in live cells, we tagged the NH2 terminus of seven full-length Rho proteins with GFP and expressed them by transient transfection in a variety of cell lines. MDCK cells (Fig 1) proved especially informative because, when grown to confluence, they assume a semicolumnar, nonoverlapping morphology such that the PM at the edge of the cell is viewed tangentially, and localization of GFP-tagged proteins in this compartment is easily scored. As we reported previously using this system, GFP-K-Ras4B (Fig 1 a) was expressed in a pattern easily distinguished from GFP-H-Ras (Fig 1 b) in that, although the former localized only to the PM, the latter localized to the PM and a discrete perinuclear structure that we have identified as Golgi (
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GFP-RhoB (Fig 1 c) localized in a pattern similar to that of GFP-H-Ras with prominent fluorescence in the PM and in a discrete perinuclear structure. Sensitivity to BFA and colocalization with mannosidase II, ßCOP (data not shown), and BODIPY ceremide C6 (Fig 2 h) confirmed that this structure was Golgi. GFP-TC10 (Fig 1 d) also localized prominently on the PM and on internal membranes, but in contrast to GFP-H-Ras and GFP-RhoB, the intracellular localization of GFP-TC10 was on cytoplasmic vesicles that colocalized with the endosomal markers LAMP (not shown) and internalized transferrin (see Fig 2 i). These motile vesicles present throughout the cell accumulated in the perinuclear region overlapping the Golgi but could be distinguished from Golgi by morphology, markers, and lack of sensitivity to BFA. Inspection of the hypervariable regions (Fig 1 j) of H-Ras, RhoB, and TC10 revealed a CXXC PM targeting motif immediately upstream of the CAAX motif. Unlike H-Ras and RhoB, TC10 has a polybasic region adjacent to the CXXC motif and thus has two potential membrane targeting motifs. The CXXC motif has been shown to be a site for dual palmitoylation in H-Ras (
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In contrast to GFP-RhoB and GFP-TC10, GFP-Rac1 was observed predominantly in the PM (Fig 1 e) in a pattern similar to that of GFP-K-Ras4B. Like K-Ras4B, Rac1 has a polybasic region immediately upstream of its CAAX motif, suggesting that this type of second signal engages a distinct trafficking pathway that does not permit steady-state accumulation on internal membranes.
Although RhoA has a polybasic region that is only one amino acid shorter than that of Rac1 (Fig 1 j), GFP-RhoA was observed almost exclusively in the cytosol, as revealed by numerous negatively imaged organelles (Fig 1 f) similar to those observed in the cytoplasm of cells expressing GFP alone. Although there was some amorphous perinuclear accumulation of GFP-RhoA, no distinct membrane structures, like those seen with GFP-RhoB, were observed. Most striking was the absence of localization of GFP-RhoA at the PM, despite levels of expression that equaled or exceeded that of the other GFP-tagged GTPases. The cytosolic localization of GFP-RhoA suggests that it is actively retained in the cytosol. Although binding to RhoGDI could account for this retention, the membrane localization of GFP-Rac1 and GFP-pCdc42hs, also known to bind RhoGDI, suggests that other factors may be involved.
To function as a PM targeting motif, the polybasic region of K-Ras4B has been shown to require a net positive charge of four or more (
To determine if these differential localizations were cell type specific, an identical analysis was performed in COS-1 (Fig 2), CHO, and ECV cells (not shown) with similar results. Although MDCK cells allowed efficient detection of PM-associated proteins, subconfluent COS-1 cells are more spread and proved superior for analyzing localization on intracellular membranes. Using COS-1 cells, the endomembrane localization of GFP-pCdc42hs (Fig 2 a) and GFP-Rac2 (Fig 2 b) was evident, with localization on the nuclear envelope, ER, and Golgi in a pattern indistinguishable from that of GFP-H-RasC181,184S (Fig 2 c), a mutant that lacks palmitoylation sites. GFP-bCdc42hs also localized predominantly on the Golgi, nuclear envelope, and ER but revealed slightly more expression in PMs and peri-Golgi vesicles (Fig 2 d). GFP-Rac1 was predominantly expressed on the PM of COS-1 cells with both peripheral and dorsal lamellipodia easily visualized (Fig 2 e) in a pattern similar to that observed for GFP-K-Ras4B (
Each GFP fusion protein, when expressed in COS-1 cells, was a substrate in vivo for prenylcysteine-directed COOH methyltransferase (pcCMT; not shown). Thus, despite the various steady-state localizations, each construct had access to the endomembrane since this is the only compartment in which pcCMT is expressed (
COS-1 cells also afforded superior views of the peri-Golgi vesicles previously reported for GFP-H-Ras (
Rho Protein Hypervariable Regions Determine Membrane Localization
To determine if, like Ras proteins, the hypervariable region of Rho GTPases contains all of the targeting information necessary to regulate localization on membranes, we tagged the isolated hypervariable regions of pCdc42hs, bCdc42hs, Rac1, and RhoA with GFP (designated GFP-GTPase-tail). GFP-pCdc42hs-tail, GFP-bCdc42hs-tail, and GFP-Rac1-tail were expressed in patterns identical to those of the full-length GFP fusion proteins (Fig 3, ac). In contrast, GFP-RhoA-tail (Fig 3 d) localized in a pattern strikingly distinct from that of the full-length protein, one that was indistinguishable from that of GFP-pCdc42hs and GFP-bCdc42hs. Inspection of the hypervariable region of RhoA (Fig 1 j) reveals a polybasic region one residue weaker than that of Rac1 and two weaker than that of K-Ras4B, such that it may not constitute a strong PM targeting motif. Thus, the hypervariable region of RhoA appears to be functionally equivalent to that of the Cdc42hs isoforms. GFP-RhoAaa73193 (Fig 3 e), a construct with a 73amino acid NH2-terminal truncation, was expressed in a pattern identical to GFP-RhoA-tail, indicating that the NH2-terminal third of the protein is required for sequestration in the cytosol either by direct involvement in proteinprotein interaction or by allowing proper folding. To further confirm that the hypervariable domains were sufficient to determine membrane localization of Rho GTPases, we constructed a GFP-tagged chimera consisting of the NH2-terminal 188 amino acids of TC10 and the COOH-terminal 20 amino acids of pCDC42hs. The localization of this construct (Fig 3 f) was distinct from that of GFP-TC10 and identical to that of GFP-pCDC42hs, confirming that the hypervariable domain is necessary and sufficient to determine membrane localization. From these data we conclude that the hypervariable domains contain all of the information required to differentially direct Rho GTPases to membrane compartments and that RhoA is unique among the Ras and Rho proteins surveyed in that it contains information in its sequence upstream of the hypervariable domain that causes it to be efficiently sequestered in the cytosol, even when overexpressed.
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To determine if the membrane localizations of the GFP fusion proteins were dependent on the geranylgeranyl modification, we constructed mutants in which the COOH-terminal L of GFP-pCdc42hs-tail and GFP-Rac1-tail were changed to M, such that the proteins would instead be modified by a farnesyl group. The localizations of the farnesylated constructs were identical to those of the geranylgeranylated versions (Fig 3g and Fig h), indicating that the length of the prenyl chain does not affect membrane targeting.
Expression Level Affects Localization of GFP-tagged Rho GTPases
The subcellular localizations described above were determined by transient transfection using the pEGFP vector that results in overexpression of the GFP-tagged protein. Although this system is informative with regard to differential membrane targeting, the results may not accurately reveal the localization of endogenous Rho GTPases because overexpression may overwhelm the capacity of endogenous RhoGDI to regulate localization. To address this problem, we established, using neomycin and cytofluorometric selection, cell lines that stably express GFP-tagged Rho proteins at four- to eightfold lower levels (determined by cytofluorimeter and immunoblot) than those observed in transient transfections (Fig 4). Like cells transiently transfected with GFP-Rac1, ECV uroepithelial cells stably expressing GFP-Rac1 (Fig 4 b) showed predominant PM localization, particularly in peripheral lamellipodia. However, unlike the transiently transfected cells, the cell lines revealed a greater proportion GFP-Rac1 in the cytosol and nucleoplasm, consistent with sequestration from membranes by binding to RhoGDI. ECV cells stably expressing GFP-K-Ras4B at similar levels revealed only PM localization (Fig 4 a), consistent with the lack of a GDI-like chaperon for K-Ras4B. PAE cells stably expressing GFP-Rac1 revealed localization in PM lamellipodia, cytosol, and nuclear envelope but exclusion from the nucleoplasm (Fig 4 c), suggesting that the nuclear localization is cell type specific. Similarly, the localization of GFP-bCdc42hs in stably transfected ECV cells paralleled that of the transiently transfected fusion protein except for considerably more cytosolic fluorescence (Fig 4 d). Both ECV (Fig 4 e) and PAE (Fig 4 f) cells stably expressing GFP-pCdc42hs revealed significantly more cytoplasmic localization than that of the transiently overexpressed GFP fusion protein. Interestingly, although GFP-pCdc42hs did not enter the nucleoplasm of ECV cells, it labeled this compartment in PAE cells (Fig 4 f), confirming the cell type dependence of nuclear localization. Finally, both ECV (Fig 4 g) and PAE (Fig 4 h) cells stably expressing GFP-RhoA reveal a pattern of localization identical to that observed by transient transfection: cytoplasmic expression with enhancement in the Golgi region without distinct membrane localization. Thus, transient overexpression of GFP-tagged Rac1 and Cdc42hs underestimates the cytosolic pool but reveals expression in the same membrane compartments observed in cells expressing levels of protein closer to endogenous.
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To validate the localization of GFP-tagged Rho GTPases, we performed indirect immunofluorescence analysis on MDCK cells (Fig 5 a). Although the morphology and resolution of membrane compartments in the fixed permeabilized cells were markedly inferior to those observed in live cells and, more important, the localization patterns were significantly affected by the method of fixation and permeabilization, this analysis had the advantage of permitting localization of endogenous proteins. RhoA was localized diffusely throughout the cytoplasm with some enhancement around the nucleus and no PM staining (Fig 5 a, i). In cells fixed with paraformaldehyde and permeabilized with Triton X-100, RhoB was observed in vesicular structures (Fig 5 a, ii) similar to those reported previously, using similar experimental conditions, as early endosomes (
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To further validate these results, we determined localization of endogenous Rho proteins and RhoGDI in COS-1 cells by subcellular fractionation (Fig 5 b). Although RhoGDI and RhoA were almost entirely soluble, 87 ± 3% of RhoB was detected in the membrane fraction, confirming both the localization of the GFP-tagged proteins and the indirect immunofluorescent localization of the endogenous counterparts. In contrast to these extremes, endogenous Rac1 and Cdc42hs were equally partitioned between soluble and membrane fractions, consistent with the capacity for both membrane and RhoGDI binding. Thus, the localization of endogenous Rho GTPases paralleled that of the corresponding GFP-tagged proteins, validating the strikingly distinct subcellular localizations of members of this family of GTPases.
Translocation of GFP-tagged Rho GTPases to the PM
Rho GTPases have been shown to translocate to membranes upon activation (
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Binding of GFP-tagged Rho GTPases to RhoGDI In Vivo
The increase in the cytosolic pool of GFP-Rac1 and GFP-Cdc42hs but not GFP-RhoA in stably transfected cell lines relative to that of transiently transfected cells suggested that the localization of these proteins was regulated by RhoGDI binding in vivo and that the buffering capacity of RhoGDI could be overcome for Rac1 and Cdc42hs but not RhoA. To test this hypothesis directly, we expressed GFP-tagged Rho GTPases at different levels with and without cooverexpression of RhoGDI (Fig 7). Although at high levels of expression, GFP-pCdc42hs, GFP-bCdc42hs, GFP-Rac1, and GFP-Rac2 were predominantly observed in membranes (Fig 7, a ii, c ii, d ii, and e ii), as described above, at lower levels of expression these proteins were largely cytosolic (Fig 7, a i, c i, d i, and e i). When these proteins were cooverexpressed at high levels with RhoGDI
, the localization was shifted to a cytosolic pattern (Fig 7, a iii, c iii, d iii, and e iii). From these data we conclude that, as expected, RhoGDI
is a regulator of Cdc42hs and Rac localization. Also, we conclude that the GFP tag at the NH2 terminus of Rho GTPases does not interfere with binding to RhoGDI
. Thus, coexpression of GFP-tagged Rho GTPases and RhoGDI
serves as a powerful assay of in vivo binding.
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We used this assay to determine if the geranylgeranyl modification rather than a farnesyl modification is required for binding to RhoGDI in vivo. We constructed a GFP-tagged pCdc42hs mutant in which the COOH-terminal L was switched to M to promote farnesylation. We confirmed that the mutant was farnesylated in our system by demonstrating that, in contrast to GFP-pCdc42hs, membrane localization of GFP-pCdc42hsL191M was blocked by a farnesyltransferase inhibitor (not shown). The expression pattern of GFP-pCdc42hsL191M was identical to that of GFP-pCdc42hs. Both low level expression and cooverexpression with RhoGDI resulted in cytosolic expression (Fig 7 b). Thus, despite the ability of the prenyl binding grove of RhoGDI
to accommodate the 20-carbon prenyl chain (
As expected, the cytosolic pattern of GFP-RhoA was altered neither by expression at low levels nor by cooverexpression of RhoGDI (Fig 7 f). The basis for the increased buffering capacity of the cytosol for GFP-RhoA relative to GFP-Rac and GFP-Cdc42hs was explored by determining the relative stoichiometry of RhoA, Rac1, Cdc42hs, and RhoGDI in three cell lines (Table 1). Although the endogenous levels of RhoGDI exceeded those of each of the three GTPases individually, the molar sum of RhoA, Rac1, and Cdc42hs was approximately equal to the molar amount of RhoGDI in each of the three cell types, suggesting coordinated regulation between the production of these GTPases and their cytosolic chaperon. Although the near unity of the RhoA plus Rac1 plus Cdc42hs/RhoGDI
molar ratio explains why overexpressing GFP-Rac and GFP-Cdc42hs leads to membrane localization, it does not explain why overexpressing GFP-RhoA does not and suggests that factors other than RhoGDI
contribute to maintaining GFP-RhoA in the cytosol.
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The fact that the molar amount of RhoGDI was roughly equivalent to that of the sum of RhoA, Rac, and Cdc42hs presented a conundrum in that it suggested that no free RhoGDI
would be available to bind other Rho family GTPases. Therefore, we employed our in vivo RhoGDI
binding assay to determine the binding capacity of two other Rho GTPases, GFP-RhoB (Fig 7 g) and GFP-TC10 (Fig 7 h). The localization of these constructs was changed neither by expression at low levels nor cooverexpression of RhoGDI
, suggesting that these proteins do not bind RhoGDI
in vivo. Thus, binding to RhoGDI
is not a universal characteristic of Rho family GTPases.
Palmitoylation Regulates Membrane Targeting and RhoGDI Binding
Recently, 2BP was reported to be an effective inhibitor of protein palmitoylation in vivo (
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To confirm that the striking difference in the localization of GFP-RhoA versus GFP-RhoB was a consequence of palmitoylation in the secondary targeting motif of RhoB, we constructed GFP-tagged RhoA/RhoB chimeras. We switched the CAAX motif alone of GFP-RhoA (CLVL) with that of RhoB (CKVL) and, as expected, found no effect on localization (not shown). In contrast, when we switched the RhoA CAAX motif with that of RhoB but included the cysteine that occurs NH2-terminal to this motif in RhoB (CCKVL), the localization of the chimera was dramatically changed to that of GFP-RhoB (Fig 8 f). Moreover, expression of this construct in the presence of 2BP converted the expression pattern to that of RhoA (Fig 8 g). Thus, a single palmitoylatable cysteine in the hypervariable domain is sufficient to overcome sequestration in the cytosol and redirect RhoA to an expression pattern similar to that of GFP-H-Ras and GFP-RhoB.
Because sequestration of RhoA in the cytosol is likely to be due, in part, to binding to RhoGDI, and because a palmitoyl group cannot be accommodated in the crystal structure of geranylgeranylated Cdc42hs bound to RhoGDI
(
binding. Cooverexpression of RhoGDI
had no effect on the RhoB-like localization of GFP-RhoA-CCKVL (Fig 8 h), and RhoGDI
could be coimmunoprecipitated with GFP-RhoA but not GFP-RhoA-CCKVL (Fig 10 d). Thus, palmitoylation blocks binding to RhoGDI
.
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To determine the role of palmitoylation in targeting TC10 to PMs and endosomes, we expressed GFP-TC10 in the absence or presence of 2BP (Fig 8i and Fig j). In the presence of the inhibitor, GFP-TC10 was mislocalized, like GFP-H-Ras and GFP-RhoB, to the nuclear envelope, ER, and Golgi. These data confirm that TC10 is palmitoylated and that palmitoylation is required for targeting to PMs and endosomes.
Differential RhoGDI Binding In Vivo of Activated and Dominant Negative Alleles of Rho GTPases
GTP binding stimulates release in vitro of Rho GTPases from RhoGDI and promotes association with membranes ( (Fig 9 a). As expected, GFP-pCdc42hs-61L, an activated allele of pCdc42hs defective in GTPase activity, was localized only in membrane compartments, and this localization was unaffected by cooverexpression of RhoGDI
. In contrast, although GFP-pCdc42hs-12V, another activated allele, when expressed alone was predominantly membrane associated, cooverexpression of RhoGDI
resulted in sequestration in the cytosol, indicating binding. Thus, although both the 61L and 12V mutants of Cdc42hs are dominant active alleles in functional assays, they differ in their capacity to bind RhoGDI
.
Dominant negative alleles of Ras-related GTPases have been thought to be locked in a GDP-bound state, despite recent evidence suggesting that they may be nucleotide-free ( (Fig 9 a). To confirm the effect of the 17N mutation on RhoGDI
binding, we examined GFP-Rac1-17N and found that it too failed to bind RhoGDI
(Fig 9 a).
To determine if the analogous mutations of RhoA would similarly affect localization, we prepared equivalent GFP-tagged constructs and localized them in live cells using confocal microscopy (Fig 9 b). As observed with epifluorescence, confocal analysis confirmed that GFP-RhoA was predominantly cytosolic as scored by diffuse fluorescence with distinct, negatively outlined organelles. In striking contrast, GFP-RhoA-14V, GFP-RhoA-63L, and GFP-RhoA-19N were all localized on both PMs and internal membranes, including the nuclear membrane and numerous vesicles. Little cytosolic fluorescence was observed. Cooverexpression of RhoGDI had no effect on the localization of GFP-RhoA-63L or GFP-RhoA-19N but induced partial relocalization of GFP-RhoA-14V to the cytosol (data not shown), concordant with the results observed for GFP-tagged pCdc42hs mutants. Thus, any of these three single amino acid changes was sufficient to release RhoA from sequestration in the cytosol, a process mediated in part by RhoGDI binding.
To verify the differential binding in vivo of Rho GTPases and their various mutants to RhoGDI as determined by fluorescence, we performed coimmunoprecipitation (Fig 10). Using this method, we observed that, although GFP-RhoA, GFP-Rac1, GFP-Rac2, GFP-pCdc42hs, and GFP-bCdc42hs were capable of binding RhoGDI
, GFP-RhoB and GFP-TC10 were not (Fig 10 a). Coimmunoprecipitation analysis of the nucleotide binding and prenylation mutants recapitulated the fluorescence assay: 12/14V mutants bound RhoGDI
but neither 61/63L nor 17/19N mutants bound to the chaperon, and the farnesylated GFP-pCdc42hsL191M bound RhoGDI
at least as well as the wild-type geranylgeranylated form (Fig 10 b).
To validate these results with a functional assay, we analyzed the effect of overexpressing RhoGDI on SRF-dependent transcriptional activation by dominant active alleles of pCdc42hs (Fig 10 c). SRF was activated more efficiently by pCdc42hs-61L than by pCdc42hs-12V, suggesting that, indeed, the former is more deficient in GTPase activity. Moreover, although pCdc42hs-12V-mediated activation was markedly inhibited by overexpression of RhoGDI
, pCdc42hs-61L-mediated activation was unaffected by RhoGDI
, confirming a lack of binding.
Coimmunoprecipitation also confirmed that the introduction of a palmitoylatable cysteine adjacent to the CAAX motif of RhoA completely abrogated RhoGDI binding and that inhibition of palmitoylation with 2BP restored binding (Fig 10 d). Finally, like TC10, the chimera consisting of 188 amino acids of TC10 fused to the 20amino acid hypervariable domain of pCdc42hs but did not bind RhoGDI
(Fig 10 d), suggesting that palmitoylation is not the only factor that prevents TC10 from binding to the chaperon. These data thereby confirm the observations that, despite a high degree of sequence homology to RhoA and Cdc42hs respectively, neither RhoB nor TC10 bind RhoGDI
and that palmitoylation inhibits binding to RhoGDI
.
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Discussion |
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The signals that target Ras proteins to membranes have been extensively characterized (, a ubiquitously expressed chaperon that has the capacity to retain COOH-terminally processed Rho proteins in the cytosol. Although prenylation has been shown to be required for binding to RhoGDI (
In this study, we used GFP fusion proteins to analyze the membrane targeting and RhoGDI binding in live cells of seven Rho family GTPases. We found that although both isoforms of Cdc42hs, Rac1, Rac2, and RhoA bind, RhoGDI, RhoB, and TC10 do not. Moreover, we found that palmitoylation of RhoA blocked RhoGDI binding and that the GTP/GDP state of those proteins that can bind RhoGDI profoundly influenced binding in vivo. When each Rho protein, except RhoA, was expressed at levels that overcame the binding capacity of RhoGDI, they localized to a variety of membranes as determined by their hypervariable domains. RhoB, like H-Ras, is palmitoylated and targeted to PMs, Golgi, and peri-Golgi vesicles. TC10, which has both a palmitoylation site and a polybasic region, is targeted to PMs and endosomes. Rac1, which has a strong polybasic region, is targeted like K-Ras4B primarily to the PM. Both isoforms of Cdc42hs and Rac2, which have weak polybasic regions, remain predominantly in the endomembrane, although some protein is expressed at the PM. Neither localization nor RhoGDI binding was affected by substituting farnesylation for geranylgeranylation of Cdc42hs. When RhoA was truncated by 73 amino acids from the NH2 terminus, it behaved like Cdc42hs, consistent with its relatively weak polybasic region, suggesting that this GTPase has an NH2-terminal domain that promotes retention in the cytosol.
Because it permits localization in live cells, the use of GFP fusion proteins to localize Rho GTPases offers several distinct advantages over previously employed methods. These include markedly superior resolution of endomembrane structures, avoidance of fixation artifacts, and the ability to directly observe dynamic localizations. The localizations of Rac1, pCdc42hs, and RhoA determined by GFP fusions were generally consistent with previous studies. Endogenous Rac1 has been localized by cell fractionation to both cytosol and crude membranes (-subunit of the coatomer complex (
COP) (
COP cycles between ERs and Golgi. Although RhoA has been reported in both cytosol and membrane pellets (
In neutrophils, Rac2 regulates assembly of the NADPH oxidase essential for host defense and, in resting cells, is in the cytosol in a 1:1 complex with RhoGDI (
Our localization of GFP-RhoB to PMs, Golgi, and peri-Golgi vesicles distinct from endosomes contrasts with the prior localization of RhoB to endosomes. Microinjected overexpressed myc-tagged RhoB was observed in paraformaldehyde-fixed cells permeabilized with Triton X-100 to be located in cytoplasmic vesicles that partially colocalized with transferrin-loaded vesicles and partially colocalized with distinct mannose 6-phosphate receptorpositive vesicles but not with Lucifer yellowlabeled vesicles (
As expected, both isoforms of Cdc42hs, Rac1, Rac2, and RhoA bound RhoGDI as determined by both the in vivo fluorescence assay and by coimmunoprecipitation. However, although Cdc42hs, Rac1, and Rac2 could be readily expressed at levels that overcame the capacity of RhoGDI to retain these molecules in the cytosol such that their various membrane localizations became evident, RhoA remained cytosolic even when expressed at very high levels. This difference is not likely due to a higher affinity of RhoA for the available endogenous pool of RhoGDI
since our measurements indicated (see Table 1) that RhoGDI
is expressed at a level equivalent to that of the sum of Cdc42hs, Rac1, and RhoA and that even if RhoA had a high enough affinity for RhoGDI
to displace bound Cdc42hs and Rac1, the total pool of RhoGDI
has only a three- to eightfold molar excess over endogenous RhoA, a deficit clearly overcome by our transient transfections. Other known isoforms of RhoGDI are not likely to account for the retention of overexpressed GFP-RhoA in the cytosol since RhoGDIß is expressed only in hematopoietic cells and has a 10-fold lower affinity for RhoA, Rac1, and Cdc42hs (
is expressed mainly in brain and pancreas, binds RhoA with low affinity, and is itself targeted to membranes by virtue of a hydrophobic NH2-terminal domain (
The failure of RhoB and TC10 to bind RhoGDI was unexpected since these GTPases are highly homologous to RhoA and Cdc42hs, respectively. Nevertheless, our data are consistent with the observation that RhoB could not be extracted from membranes by RhoGDI
(
in complex with geranylgeranylated Cdc42hs reveals a hydrophobic binding pocket for the geranylgeranyl isoprenoid (
. However, this could not explain entirely the lack of binding of RhoB and TC10, since mutants that lacked palmitoylation sites did not bind RhoGDI
. The proteinprotein interface determined by the cocrystalization of Cdc42hs and RhoGDI
revealed that amino acids 103, 104, 184, and 186 of Cdc42hs (HHKR) form hydrogen bonds with specific residues of RhoGDI
(
suggests that the trafficking of RhoB and TC10 is likely distinct from that of the other members of the Rho family and perhaps similar to that of Ras proteins.
The reversal of relative affinities for RhoGDI versus membranes that we observed in dominant active mutations of RhoA and Cdc42hs is consistent with previous studies of the capacity of RhoGDI
to extract the GDP-bound but not the GTP-bound form of Rho proteins from membranes (
, RhoA14V and pCdc42hs12V retain some RhoGDI binding capacity, is consistent with the hypothesis that the former, like the Rap1 Q61 mutant, is GTPase-dead whereas the latter, like Rap1 G12 mutants, is sensitive to GAP and intrinsic GTPase activity; therefore RhoGDI
can bind the GDP-bound pool and sequester molecules in the inactive state. Alternatively, since in vitro GTP-bound Cdc42hs binds to RhoGDI
as well as the GDP-bound form (
The membrane localization of 17/19N dominant negative mutants of RhoA, pCdc42hs, and Rac1 and their failure to bind RhoGDI was unexpected since these molecules have been thought to be locked in a GDP-bound state that would be expected to have high affinity for RhoGDI
. However, recent evidence suggests that rather than existing in a GDP-bound state in complex with RhoGDI
, RhoA-19N is nucleotide-free and does not coprecipitate with RhoGDI
(
, the lack of binding to RhoGDI
of 17/19N mutants and the resulting membrane localization may play a significant role in their mode of action, e.g., bringing them into proximity with membrane-associated GEFs. Alternatively, since most Dbl family GEFs are believed to function on membranes, it is possible that the membrane localization of 17/19N mutants and their inability to bind RhoGDI
in vivo are both mediated by high affinity binding to membrane-associated Dbl family proteins. In either case, the previously unappreciated endomembrane localization of 17/19N mutants that we observed is likely to play a significant role in their mechanism of action.
The most striking result of our analysis of GFP-tagged Rho proteins in live cells is the diversity of localizations despite a common pathway of posttranslational modification. Our data indicate that these localizations are regulated primarily by determinants in the hypervariable region upstream of the CAAX motif and by the capacity to bind RhoGDI. However, the relatively subtle differences in localization observed between proteins with very similar hypervariable regions and RhoGDI binding capacities, e.g., pCdc42hs versus Rac2, suggest that other factors can influence localization. The diversity of localization matches the diversity of function reported for the Rho family of proteins. In some cases localization suggests function. For example, the ER and Golgi localization of pCdc42hs is consistent with its ability to bind COP and regulate ER to Golgi transport (
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Footnotes |
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The online version of this article contains supplemental material.
1 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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Acknowledgements |
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We thank Arie Abo, Gary Bokoch, Richard Cerione, Adrienne Cox, Alan Hall, and Danny Manor for providing cDNAs. We thank Marianne Feoktistov and Pheobe Recht for expert technical assistance.
This work was supported by National Institutes of Health research grants GM55279, AI36224, General Clinical Research Center grant M01 RR00096 and training grant T32 GM07308; The Burroughs Wellcome Fund; and the National Science Foundation Major Instrumentation Award 9977430.
Submitted: 4 August 2000
Revised: 21 November 2000
Accepted: 29 November 2000
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References |
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