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• W1974273182 abstract "The COOH-terminal polybasic region (PBR) of Rac1, a Rho family GTPase member, is required for Rac1 self-association, membrane localization, nuclear translocation, and interaction with downstream effectors. We previously demonstrated that phosphatidylinositol-4-phosphate 5-kinase, one of the effectors that requires the polybasic region for interaction, is necessary for efficient invasin-promoted uptake of Yersinia pseudotuberculosis by nonphagocytic cells. Here we further examined the role of this region in invasin-promoted uptake. Using fluorescence resonance energy transfer experiments (FRET), we determined that engagement of integrin receptors by invasin caused elevated levels of Rac1 self-association at the site of bacterial adhesion in a PBR-dependent fashion. Self-association could be disrupted using several strategies: translocation of the Yersinia YopT prenylcysteine protease into host cells, inactivation of the Rac1 isoprenylation signal that is required for membrane localization, and elimination of the PBR. Disruption in each case impaired invasin-promoted uptake. To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail. The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association. This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors. The COOH-terminal polybasic region (PBR) of Rac1, a Rho family GTPase member, is required for Rac1 self-association, membrane localization, nuclear translocation, and interaction with downstream effectors. We previously demonstrated that phosphatidylinositol-4-phosphate 5-kinase, one of the effectors that requires the polybasic region for interaction, is necessary for efficient invasin-promoted uptake of Yersinia pseudotuberculosis by nonphagocytic cells. Here we further examined the role of this region in invasin-promoted uptake. Using fluorescence resonance energy transfer experiments (FRET), we determined that engagement of integrin receptors by invasin caused elevated levels of Rac1 self-association at the site of bacterial adhesion in a PBR-dependent fashion. Self-association could be disrupted using several strategies: translocation of the Yersinia YopT prenylcysteine protease into host cells, inactivation of the Rac1 isoprenylation signal that is required for membrane localization, and elimination of the PBR. Disruption in each case impaired invasin-promoted uptake. To determine if there is a role for the PBR in Rac1 effector signaling that was independent of its role in membrane localization or multimerization, we examined the effect of the PBR in the context of a Rac1 derivative that was targeted to the membrane via an NH2-terminal lipid tail. The membrane-targeted Rac1 derivative restored significant invasin-promoted bacterial uptake in a PBR-dependent manner and yet displayed no detectable self-association. This study indicates that, in addition to its role in promoting membrane localization, the PBR exerts a positive effect on Rac1-controlled bacterial uptake that is independent of Rac1 self-association, most likely due to signaling to downstream effectors. Uptake of pathogenic bacteria by normally nonphagocytic cells is uniformly regulated by members of the Rho GTPase family, including Cdc42, Rac1, and RhoA (1Gruenheid S. Finlay B.B. Nature. 2003; 422: 775-781Crossref PubMed Scopus (252) Google Scholar). In the case of the Gram-negative enteropathogenic bacterium Yersinia pseudotuberculosis, Rac1 is required for uptake, whereas Cdc42 and RhoA play either no role or a negative role, respectively (2Alrutz M.A. Srivastava A. Wong K.W. D'Souza-Schorey C. Tang M. Ch'Ng L.E. Snapper S.B. Isberg R.R. Mol. Microbiol. 2001; 42: 689-703Crossref PubMed Scopus (85) Google Scholar, 3Black D.S. Bliska J.B. Mol. Microbiol. 2000; 37: 515-527Crossref PubMed Scopus (248) Google Scholar, 4McGee K. Zettl M. Way M. Fallman M. FEBS Lett. 2001; 509: 59-65Crossref PubMed Scopus (44) Google Scholar). Rac1 can facilitate bacterial uptake by remodeling the actin cytoskeleton through one of three mechanisms: 1) inducing actin filament nucleation and branching by activating the Arp2/3 complex via WAVE family members (5Takenawa T. Miki H. J. Cell Sci. 2001; 114: 1801-1809Crossref PubMed Google Scholar); 2) increasing phosphoinositol 4,5-bisphosphate concentrations in the plasma membrane, resulting in uncapping of actin filaments (6Wong K.W. Isberg R.R. J. Exp. Med. 2003; 198: 603-614Crossref PubMed Scopus (72) Google Scholar, 7Tolias K.F. Hartwig J.H. Ishihara H. Shibasaki Y. Cantley L.C. Carpenter C.L. Curr. Biol. 2000; 10: 153-156Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar); or 3) inhibiting actin depolymerization by activating LIM kinases, which deactivate cofilin (8Bierne H. Gouin E. Roux P. Caroni P. Yin H.L. Cossart P. J. Cell Biol. 2001; 155: 101-112Crossref PubMed Scopus (156) Google Scholar). Activation of Rac1 requires GTP loading by guanine nucleotide exchange factors (RacGEFs), which show specificity for subclasses of Rho family members (9Rossman K.L. Der C.J. Sondek J. Nat. Rev. Mol. Cell. Biol. 2005; 6: 167-180Crossref PubMed Scopus (1327) Google Scholar). Exchange takes place simultaneously with release of Rac1 from RhoGDI proteins, which maintain Rho family GTPases in an inactive state in the cell cytoplasm (10Dovas A. Couchman J.R. Biochem. J. 2005; 390: 1-9Crossref PubMed Scopus (321) Google Scholar). Release allows insertion of Rac1 in a target membrane via a prenyl group linked to the carboxyl terminus of the protein (11Robbe K. Otto-Bruc A. Chardin P. Antonny B. J. Biol. Chem. 2003; 278: 4756-4762Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). After exchange and insertion into the membrane, active Rac1 is able to bind downstream effectors, many of which modulate the actin dynamics associated with bacterial uptake (12Ridley A.J. Trends Cell Biol. 2006; 16: 522-529Abstract Full Text Full Text PDF PubMed Scopus (891) Google Scholar). The activation observed is often a response to engagement of cell surface receptors, resulting in interaction with downstream effectors (13del Pozo M.A. Price L.S. Alderson N.B. Ren X.D. Schwartz M.A. EMBO. 2000; 19: 2008-2014Crossref PubMed Scopus (405) Google Scholar, 14Del Pozo M.A. Kiosses W.B. Alderson N.B. Meller N. Hahn K.M. Schwartz M.A. Nat. Cell Biol. 2002; 4: 232-239Crossref PubMed Scopus (293) Google Scholar). One example of a group of host cell surface molecules that activate Rac1 in response to substrate engagement is the β1 integrin receptor family, the members of which bind envelope proteins encoded by a wide range of pathogenic microorganisms (15Rottner K. Stradal T.E. Wehland J. Dev. Cell. 2005; 9: 3-17Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 16Stewart P.L. Nemerow G.R. Trends Microbiol. 2007; 15: 500-507Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Y. pseudotuberculosis undergoes high efficiency bacterial uptake after engagement of β1 integrin receptors by the bacterial cell surface protein invasin (17Isberg R.R. Voorhis D.L. Falkow S. Cell. 1987; 50: 769-778Abstract Full Text PDF PubMed Scopus (411) Google Scholar). Invasin binds integrins with a much higher affinity than natural ligands, such as fibronectin and laminin (18Van Nhieu G.T. Isberg R.R. J. Biol. Chem. 1991; 266: 24367-24375Abstract Full Text PDF PubMed Google Scholar). Invasin is also able to form multimers, which is predicted to allow receptor clustering, thought to be a prerequisite for triggering intracellular signaling processes required for bacterial uptake (19Dersch P. Isberg R.R. EMBO J. 1999; 18: 1199-1213Crossref PubMed Scopus (115) Google Scholar). The combined activities of high affinity binding and multimerization by invasin are critical for high efficiency invasin-mediated bacterial uptake that is regulated by activated Rac1 (19Dersch P. Isberg R.R. EMBO J. 1999; 18: 1199-1213Crossref PubMed Scopus (115) Google Scholar, 20Leong J.M. Morrissey P.E. Marra A. Isberg R.R. EMBO J. 1995; 14: 422-431Crossref PubMed Scopus (69) Google Scholar). Engagement of β1 integrins by Y. pseudotuberculosis triggers efficient recruitment of Rac1 to nascent phagosomal membranes, resulting in localized accumulation of the activated GTPase, as determined by FRET 4The abbreviations used are:FRETfluorescence resonance energy transferPBRpolybasic regionCFPcyan fluorescent proteinmCFPmonomeric CFPYFPyellow fluorescent proteinmYFPmonomeric YFPWTwild typeGAPGTPase-activating proteinHAhemagglutininPIP5Kphosphatidylinositol-4-phosphate 5-kinase analysis (2Alrutz M.A. Srivastava A. Wong K.W. D'Souza-Schorey C. Tang M. Ch'Ng L.E. Snapper S.B. Isberg R.R. Mol. Microbiol. 2001; 42: 689-703Crossref PubMed Scopus (85) Google Scholar). Although the most attractive model for Rac1 function at the phagocytic cup is that localized activation of Rac1 occurs at sites of receptor engagement, it is possible that active Rac1 is simply delivered to these sites by release of the GTP-loaded form from their soluble RhoGDI-bound complexes in the host cell cytoplasm. The latter possibility was suggested from a study in which fibronectin-coated beads were used to challenge cultured cells (14Del Pozo M.A. Kiosses W.B. Alderson N.B. Meller N. Hahn K.M. Schwartz M.A. Nat. Cell Biol. 2002; 4: 232-239Crossref PubMed Scopus (293) Google Scholar). This raises the possibility that Rac1-GTP can be sequestered by cytosolic RhoGDI and then directly delivered to the site of receptor clustering without a membrane-dependent activation step. fluorescence resonance energy transfer polybasic region cyan fluorescent protein monomeric CFP yellow fluorescent protein monomeric YFP wild type GTPase-activating protein hemagglutinin phosphatidylinositol-4-phosphate 5-kinase An additional mechanism for regulating the activity of Rac1 has been proposed. Gel filtration studies and co-immunoprecipitation experiments indicated that the polybasic region (PBR) at the COOH terminus of Rac1 mediates self-association of Rac1 (21Zhang B. Gao Y. Moon S.Y. Zhang Y. Zheng Y. J. Biol. Chem. 2001; 276: 8958-8967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). This self-association is independent of the nucleotide status of Rac1. It has been suggested that PBR-mediated self-association potentiates Rac1-GTP to activate effectors, based on the observation that Rac1 derivatives lacking the PBR are defective for activation of the serine/threonine kinase PAK1 (21Zhang B. Gao Y. Moon S.Y. Zhang Y. Zheng Y. J. Biol. Chem. 2001; 276: 8958-8967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). If local engagement of β1 integrin receptors indeed triggers a localized RhoGDI release from Rac1, there should also be an induction of Rac1 self-association at the sites of integrin engagement. In this report, we investigate the role of the PBR in supporting invasin-mediated uptake of Y. pseudotuberculosis and identify sequence elements that are important for Rac1 self-association. Using derivatives that allow membrane localization of Rac1 without the presence of the PBR, we provide evidence that the role of this sequence in the uptake process appears to be independent of its role in self-association, presumably because the PBR is necessary to interact with downstream effectors or guanine nucleotide exchange factors. Cell Culture, Transfection, and Plasmid Constructs—Culture and transfection of COS1 cells were performed as previously described (2Alrutz M.A. Srivastava A. Wong K.W. D'Souza-Schorey C. Tang M. Ch'Ng L.E. Snapper S.B. Isberg R.R. Mol. Microbiol. 2001; 42: 689-703Crossref PubMed Scopus (85) Google Scholar). Mammalian expression plasmids pmCFP-Rac1 and pmYFP-Rac1 encoding the NH2-terminal fusion of monomeric cyan fluorescence protein (CFP) or yellow fluorescence protein (YFP) to Rac1 as well as Rac1 derivatives having the G12V, R66A, C189S, or 6Q (183KKRKRK → 183QQQQQQ) mutations have been described (22Wong K.W. Isberg R.R. PLoS Pathog. 2005; 1: 125-136Crossref Scopus (57) Google Scholar, 23Wong K.W. Mohammadi S. Isberg R.R. J. Biol. Chem. 2006; 281: 40379-40388Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The K186E mutation in Rac1 was generated using the Stratagene (La Jolla, CA) QuikChange site-directed mutagenesis kit. pLyn-mCFP consists of a 10-amino acid myristoylation/palmitoylation sequence of Lyn kinase fused to the 5′-end of the mCFP gene (6Wong K.W. Isberg R.R. J. Exp. Med. 2003; 198: 603-614Crossref PubMed Scopus (72) Google Scholar). pLyn-mYFP was generated by replacing mCFP with mYFP. Rac1(C189S) or Rac1(6Q/C189S) devoid of the geranylgeranylation signal were inserted in frame into the COOH terminus of pLyn-mCFP to generate pLyn-mCFP-Rac1(C189S) or pLyn-mCFP-Rac1(6Q/C189S). mCFP-GerGer and mYFP-GerGer containing fluorescence protein fused to the CAAX geranylgeranylation signals, without the upstream polybasic region, were kindly provided by Dr. R. Tsien (University of California, San Diego) (24Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1803) Google Scholar). Plasmids encoding HA-mYFP, Lyn-HA-mYFP, Myc-mCFP, and Lyn-Myc-mCFP fusions were constructed by replacing the enhanced green fluorescent protein gene in pEGFP-C1 (Clontech) with each tag-encoded gene indicated. Various Rac1 alleles (WT, R66A, 6Q, C189S, and 6Q/C189S) were then cloned into all four plasmids. All plasmids were verified by sequencing. Oligonucleotide sequences are available upon request. Rac1 derivatives used in this study and their properties are described in Table 1.TABLE 1Rac1 derivatives used in this studyConstructionsPropertiesPredicted localizationmCFP-Rac1 (WT)Wild typeCytoplasm and membranemYFP-Rac1 (WT)Wild typeCytoplasm and membranemYFP-Rac1 (6Q)Missing Rac1 PBRCytoplasmmCFP-Rac1 (G12V)GTP hydrolysis-defectiveCytoplasm and membranemCFP-Rac1 (R66A)Defective for RhoGDI bindingPredominantly membranemCFP-Rac1 (C189S)Missing prenylation siteNucleus and cytoplasmmYFP-Rac1 (K186E)Defective for PIP5K bindingMembrane and cytoplasmmCFP-CAAXPrenylated mCFPMembranemYFP-CAAXPrenylated mYFPMembranemCFP-Rac1 (G12V) (6Q)GTP hydrolysis-defective; missing PBRCytoplasmmCFP-Rac1 (G12V) (C189S)GTP hydrolysis defective; missing prenylation siteCytoplasm and nucleusmCFP-Rac1 (G12V) (K186E)GTP hydrolysis-defective; defective for PIP5K bindingMembrane and cytoplasmLyn-mCFP-Rac1 (6Q) (C189S)Myristoylated; missing PBR and prenylation siteMembraneLyn-mCFP-Rac1 (C189S)Myristoylated; missing prenylation siteMembraneLyn-mYFP-Rac1 (C189S)Myristoylated; missing prenylation siteMembraneLyn-mCFP-Rac1 (G12V) (K186E)Myristoylated; GTP hydrolysis-defective; defective for PIP5K bindingMembraneLyn-mYFP-Rac1 (G12V) (K186E)Myristoylated; GTP hydrolysis-defective; defective for PIP5K bindingMembraneLyn-mYFP-Rac1 (G12V) (C189S)Myristoylated; GTP hydrolysis-defective; missing prenylation siteMembraneLyn-mCFP-Rac1 (G12V) (C189S)Myristoylated; GTP hydrolysis defective; missing prenylation siteMembraneLyn-mCFP-Rac1 (6Q) (C189S)Myristoylated; missing PBR and prenylation siteMembraneLyn-mYFP-Rac1 (6Q) (C189S)Myristoylated; missing PBR and prenylation siteMembraneLyn-mCFP-Rac1 (G12V) (6Q) (C189S)Myristoylated; GTP hydrolysis-defective; missing PBR and prenylation siteMembraneLyn-mYFP-Rac1 (G12V) (6Q) (C189S)Myristoylated; GTP hydrolysis-defective; missing PBR and prenylation siteMembraneMyc-mCFP-Rac1 (WT)Wild typeCytoplasm and membraneHA-mYFP-Rac1 (WT)Wild typeCytoplasm and membraneMyc-mCFP-Rac1 (6Q)Missing PBRCytoplasmMyc-mCFP-Rac1 (C189S)GTP hydrolysis-defective; missing prenylation siteCytoplasm and nucleusMyc-mCFP-Rac1 (R66A)Defective for RhoGDI bindingPredominantly membraneLyn-Myc-mCFP-Rac1 (6Q)Myristoylated; missing PBRMembraneLyn-HA-mYFP-Rac1 (6Q)Myristoylated; missing PBRMembraneLyn-HA-mYFP-Rac1 (C189S)Myristoylated; missing prenylation siteMembraneLyn-Myc-mCFP-Rac1 (C189S)Myristoylated; missing prenylation siteMembraneLyn-HA-mYFP-Rac1 (6Q) (C189S)Myristoylated; missing PBR; missing prenylation siteMembraneLyn-Myc-mCFP-Rac1 (6Q) (C189S)Myristoylated; missing PBR and prenylation siteMembraneLyn-Myc-mCFPMyristoylated mCFPMembraneLyn-HA-mYFPMyristoylated mYFPMembrane Open table in a new tab Culture of Y. pseudotuberculosis Infection of Mammalian Cells and Immunofluorescence Protection Assay of Bacterial Uptake—Conditions for growth of virulence plasmid-cured Y. pseudotuberculosis YPIII(p-) with or without YopE or YopT and infection of COS1 have been described (22Wong K.W. Isberg R.R. PLoS Pathog. 2005; 1: 125-136Crossref Scopus (57) Google Scholar). The plasmid-cured Y. pseudotuberculosis strain lacks the virulence plasmid (pYV) that encodes YopE and YopT and is efficiently internalized into host cells. Strains that harbor the YopE Rho family GAP (22Wong K.W. Isberg R.R. PLoS Pathog. 2005; 1: 125-136Crossref Scopus (57) Google Scholar) or the YopT family CAAX protease (kind gift of Dr. James Bliska, SUNY Stony Brook) contain plasmids encoding these proteins as well as the plasmid pYV (yopT-deficient yopE::kan yopH::cam; referred to as strain YP17) to allow translocation of these proteins via the bacterial type III secretion system (22Wong K.W. Isberg R.R. PLoS Pathog. 2005; 1: 125-136Crossref Scopus (57) Google Scholar). For bacteria lacking the virulence plasmid, the YPIII(p-) strain was grown logarithmically in Luria Bertani broth at 26 °C until an A600 of 0.7, prior to inoculation onto cultured mammalian cells (22Wong K.W. Isberg R.R. PLoS Pathog. 2005; 1: 125-136Crossref Scopus (57) Google Scholar). For strains harboring the pYV plasmid and Yop-encoding plasmids, bacteria were grown with aeration at 26 °C overnight in broth supplemented with 2.5 mm CaCl2 and 100 μg/ml ampicillin and then subcultured and grown at 26 °C until A600 of 0.2. At this point, the cultures were shifted to 37 °C and aerated for 1 h. A multiplicity of infection of 50:1 was used for YPIII(p-) incubations, and a multiplicity of infection of 25:1 was used for other derivatives. For the pYopE-expressing plasmid, 0.1 mm isopropyl-β-d-thiogalactopyranoside was supplemented during infection to induce YopE expression. Bacterial uptake was assayed using immunofluorescence protection as described (6Wong K.W. Isberg R.R. J. Exp. Med. 2003; 198: 603-614Crossref PubMed Scopus (72) Google Scholar). Briefly, bacteria appropriately cultured were incubated with transfected cells for 30 min at a multiplicity of infection of 50 at 37 °C. After incubation, the adherent cells were analyzed for internalized or surface-bound bacteria as described previously (6Wong K.W. Isberg R.R. J. Exp. Med. 2003; 198: 603-614Crossref PubMed Scopus (72) Google Scholar). The monolayers were fixed in 3! paraformaldehyde and probed with primary antibody directed against Y. pseudotuberculosis, followed by a fluorescent secondary antibody (anti-IgG conjugated to either Alexa Fluor 594 or Cascade Blue) to detect extracellular bacteria. The cells were then permeabilized (2Alrutz M.A. Srivastava A. Wong K.W. D'Souza-Schorey C. Tang M. Ch'Ng L.E. Snapper S.B. Isberg R.R. Mol. Microbiol. 2001; 42: 689-703Crossref PubMed Scopus (85) Google Scholar) and probed with antibodies directed against the bacteria to allow detection of both intracellular and extracellular bacteria. The coverslips were then probed with appropriate secondary antibodies to detect intracellular bacteria. FRET Measurements—The basis of the assay is that association between Rac1 derivatives fused to either monomeric CFP or monomeric YFP should be detected as a FRET readout. COS1 cells were first transfected with a combination of various derivatives of mCFP-Rac1 and mYFP-Rac1 and then challenged with bacteria. Infections were stopped by fixation after 30 min. The cells were then imaged and analyzed for sensitized FRET from CFP to YFP essentially as described (22Wong K.W. Isberg R.R. PLoS Pathog. 2005; 1: 125-136Crossref Scopus (57) Google Scholar), using correction factors for CFP (0.32) and YFP (0.18) for bleed-through from CFP emission and cross-YFP excitation by the FRET filter set. To measure FRET, images from YFP, CFP, and FRET filter sets (22Wong K.W. Isberg R.R. PLoS Pathog. 2005; 1: 125-136Crossref Scopus (57) Google Scholar) were captured, choosing regions of interest about nascent phagosomes. Sensitized FRET was then calculated from these regions, by subtracting the CFP and YFP correction factors, using exactly the same procedure as described previously (22Wong K.W. Isberg R.R. PLoS Pathog. 2005; 1: 125-136Crossref Scopus (57) Google Scholar). FRET signals were normalized by combination of mCFP and mYFP emissions calculated from the CFP and YFP filter sets using the following formula as described (25Xia Z. Liu Y. Biophys. J. 2001; 81: 2395-2402Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). NormalizedFRET=sensitizedFRETCFP×YFP(Eq. 1) Determination of mYFP-Rac1 Expression Levels Relative to Endogenous Rac1—COS1 cells were transfected with mYFP-Rac1 and cultured overnight. Transfected cells were lifted and subjected to flow cytometry using YFP fluorescence to sort cells into four fractions according to levels of fluorescence (YFP negative, low, medium, and high). A portion of the sorted cells were plated onto fibronectin-coated coverslips, allowed to adhere for ∼3 h, fixed in 4! paraformaldehyde, and imaged to quantify YFP fluorescence. YFP fluorescence quantification was performed as described above for FRET experiments. The remaining sorted cells were lysed in sample buffer; lysates were resolved by SDS-PAGE, blotted, and probed for Rac1 using a monoclonal anti-Rac1 antibody (clone 23A8; Sigma). Immunoprecipitation of Myc/HA-tagged mCFP/mYFP Fusions—293T cells were transfected with 0.5 μg of each plasmid in 6-well dishes. Bait constructs were Myc-tagged, and prey constructs were HA-tagged. Cells were lysed 24 h post-transfection in 500 μl of lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1! Nonidet P-40, and complete protease inhibitor mixture (Roche Applied Science)). Lysates were then incubated at 4 °C (with agitation) for 10 min and spun to remove debris. 50 μl of the cleared lysates was saved as the input fraction, and 400 μl was applied to washed anti-HA epitope affinity resin (monoclonal anti-HA; Sigma) and incubated at 4 °C (with agitation) for 1 h. The resin was washed three times in wash buffer (25 mm Tris, pH 7.5, 30 mm MgCl2, 40 mm NaCl, 0.1! Nonidet P-40), and bound proteins were eluted in 100 μl of sample buffer. 10 μl of eluted proteins (immunoprecipitate) as well as 10 μl of cleared lysates (input) were analyzed by SDS-PAGE and Western blotting. Input and immunoprecipitated proteins were detected using an anti-Myc epitope antibody (rabbit polyclonal; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as well as an anti-HA epitope antibody (rabbit polyclonal; Santa Cruz Biotechnology). Rac1 Self-associates in Cultured Cells—Y. pseudotuberculosis employs a variety of proteins to activate and misregulate Rac1 in host cells (22Wong K.W. Isberg R.R. PLoS Pathog. 2005; 1: 125-136Crossref Scopus (57) Google Scholar). In this report, we examined whether Y. pseudotuberculosis could also control self-association of Rac1 (21Zhang B. Gao Y. Moon S.Y. Zhang Y. Zheng Y. J. Biol. Chem. 2001; 276: 8958-8967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Previously, self-association was demonstrated in vitro by gel filtration chromatography and immunoprecipitation of differentially tagged Rac1 derivatives in an event requiring the COOH-terminal PBR (Fig. 1A) (21Zhang B. Gao Y. Moon S.Y. Zhang Y. Zheng Y. J. Biol. Chem. 2001; 276: 8958-8967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). To determine if self-association occurs in cells and can be detected at spatially distinct sites within the cell, Rac1 self-association was analyzed by a FRET-based system, using monomeric CFP and YFP derivatives (mCFP-Rac1 and mYFP-Rac1) (Fig. 1C). When coexpressed in the same cells, mCFP-Rac1(WT) and mYFP-Rac1(WT) produced FRET throughout the cells in all regions except for the nucleus (Fig. 1, B and C). When normalized to the concentration of Rac1 at individual sites in the cell, FRET signals (normalized FRET) were notably stronger at membrane ruffles, indicating enhanced Rac1 self-association (Fig. 1C, arrows). These elevated FRET levels were specific to these sites, because at regions where no ruffles were evident, the lower levels of normalized FRET were independent of Rac1 concentrations. Consistent with biochemical data, self-association of Rac1, as detected by FRET, required the presence of the PBR on both partners. Co-transfection of mCFP-Rac1(WT) with mYFP-Rac1(6Q), which has each of the basic residues in the PBR replaced with Gln (26Knaus U.G. Wang Y. Reilly A.M. Warnock D. Jackson J.H. J. Biol. Chem. 1998; 273: 21512-21518Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), produced a lower FRET signal (Fig. 1, B and D; 6Q), confirming the previous published findings that the PBR mediates Rac1 self-association in vitro (21Zhang B. Gao Y. Moon S.Y. Zhang Y. Zheng Y. J. Biol. Chem. 2001; 276: 8958-8967Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Self-association Is Not Due to Overexpression of Rac1 Constructs—Since any self-association observed by FRET could result from aggregation of overexpressed protein, the concentration of Rac1 expressed from the transfected plasmids relative to endogenous levels of Rac1 was determined. COS1 cells were transfected with mYFP-Rac1 and sorted by flow cytometry to determine the concentration of Rac1 relative to the amount of YFP fluorescence observed by microscopy. The transfected cells showed a broad distribution of fluorescence (Fig. 2A), with a peak of untransfected cells and a shoulder of cells having increasing amounts of fluorescence. The total cell population was collected into four separate fractions consisting of cells with increasing amounts of YFP fluorescence (neg, lo, med, hi; Fig. 2A), and each population was analyzed to determine the amount of mYFP-Rac1 expression as well as the level of fluorescence using our standard microscopic techniques. The expression of mYFP-Rac1 in the YFP-lo fraction was lower than that of endogenous Rac1, based on Western blotting with anti-Rac1 antibody. The levels of mYFP-Rac1 were about 50! of the levels of endogenous Rac1 in this population (Fig. 2B; YFP lo). In contrast, the other sorted fractions (Fig. 2B; YFP med and YFP hi) expressed much higher levels of YFP-Rac1 compared with endogenous Rac1. Transfectants plated onto coverslips from the YFP-lo population clearly had fluorescence levels similar to those being used for FRET analysis (Fig. 2, C and D; compare lo versus med). When the fluorescence was determined on individual cells from the mYFP-lo fraction, the average intensity of individual cells was ∼500 fluorescence units (Fig. 2D), which is about the mean intensity of all the cells used for FRET (Fig. 2D; mYFP-Rac1(FRET)). In fact, the cell populations that showed overexpression of YFP-Rac1 gave fluorescence intensities that were well beyond what was analyzed by FRET (Fig. 2D; med). Therefore, a large proportion of the cells analyzed by FRET expressed amounts of mYFP-Rac1 that are at or below endogenous levels of Rac1. We conclude that self-association is not due to overexpression of the fluorescence derivatives. Plasma Membrane Localization of Rac1 Is Not Sufficient to Promote Self-association—The 6Q mutation that replaces the PBR has the secondary effect of interfering with plasma membrane localization of Rac1 (14Del Pozo M.A. Kiosses W.B. Alderson N.B. Meller N. Hahn K.M. Schwartz M.A. Nat. Cell Biol. 2002; 4: 232-239Crossref PubMed Scopus (293) Google Scholar) (Fig. 3, compare A with E). We therefore tested whether blocking plasma membrane localization would affect the ability of Rac1 to self-associate. The C189S mutation, immediately downstream of PBR, prevents geranylgeranylation of Rac1 at the carboxyl-terminal CAAX box and resulted in a large pool of the protein localizing in the nucleus, as previously described (27Kreck M.L. Freeman J.L. Abo A. Lambeth J.D. Biochemistry. 1996; 35: 15683-15692Crossref PubMed Scopus (92) Google Scholar) (Fig. 3C). This lipid modification is essential for the Ras superfamily of small GTPases to anchor onto the plasma membrane (28Hancock J.F. Paterson H. Marshall C.J. Cell. 1990; 63: 133-139Abstract Full Text PDF PubMed Scopus (851) Google Scholar). Based on the FRET assay, mCFP-Rac1(C189S) showed no ability to interact with mYFP-Rac1(WT) (Fig. 1, B and E; C189S), indicating the importance of plasma membrane localization for Rac1 self-association. The C189S mutation also blocks RhoGDI binding (29Michaelson D. Silletti J. Murphy G. D'Eustachio P. Rush M. Philips M.R. J. Cell Biol. 2001; 152: 111-126Crossref PubMed Scopus (574) Google Scholar), raising the possibility that RhoGDI could play some role in Rac1 self-association. To rule out this possibility, the interaction of the Rac1(R66A) mutant was analyzed, which is unable to bind RhoGDI but maintains plasma membrane localization (22Wong K.W. Isberg R.R. PLoS Pathog. 2005; 1: 125-136Crossref Scopus (57) Google Scholar, 30Gibson R.M. Wilson-Delfosse A.L. Biochem. J. 2001; 359: 285-294Crossref PubMed Scopus (29) Google Scholar). mCFP-Rac1(R66A) p" @default.
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