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The Single Transmembrane Segment Drives Self-assembly of OutC and the Formation of a Functional Type II Secretion System in Erwinia chrysanthemi

2006; Elsevier BV; Volume: 281; Issue: 44 Linguagem: Inglês

10.1074/jbc.m606245200

1083-351X

Frédéric H. Login, Vladimir E. Shevchik,

Bacteriophages and microbial interactions

Many pathogenic Gram-negative bacteria secrete toxins and lytic enzymes via a multiprotein complex called the type II secretion system. This system, named Out in Erwinia chrysanthemi, consists of 14 proteins integrated or associated with the two bacterial membranes. OutC, a key player in this process, is probably implicated in the recognition of secreted proteins and signal transduction. OutC possesses a short cytoplasmic sequence, a single transmembrane segment (TMS), and a large periplasmic region carrying a putative PDZ domain. A hydrodynamic study revealed that OutC forms stable dimers of an elongated shape, whereas the PDZ domain adopts a globular shape. Bacterial two-hybrid, cross-linking, and pulldown assays revealed that the self-association of OutC is driven by the TMS, whereas the periplasmic region is dispensable for self-association. Site-directed mutagenesis of the TMS revealed that cooperative interactions between three polar residues located at the same helical face provide adequate stability for OutC self-assembly. An interhelical H-bonding mediated by Gln29 appears to be the main driving force, and two Arg residues located at the TMS boundaries are essential for the stabilization of OutC oligomers. Stepwise mutagenesis of these residues gradually diminished OutC functionality and self-association ability. The triple OutC mutant R15V/Q29L/R36A became monomeric and nonfunctional. Self-association and functionality of the triple mutant were partially restored by the introduction of a polar residue at an alternative position in the interhelical interface. Thus, the OutC TMS is more than just a membrane anchor; it drives the protein self-association that is essential for formation of a functional secretion system. Many pathogenic Gram-negative bacteria secrete toxins and lytic enzymes via a multiprotein complex called the type II secretion system. This system, named Out in Erwinia chrysanthemi, consists of 14 proteins integrated or associated with the two bacterial membranes. OutC, a key player in this process, is probably implicated in the recognition of secreted proteins and signal transduction. OutC possesses a short cytoplasmic sequence, a single transmembrane segment (TMS), and a large periplasmic region carrying a putative PDZ domain. A hydrodynamic study revealed that OutC forms stable dimers of an elongated shape, whereas the PDZ domain adopts a globular shape. Bacterial two-hybrid, cross-linking, and pulldown assays revealed that the self-association of OutC is driven by the TMS, whereas the periplasmic region is dispensable for self-association. Site-directed mutagenesis of the TMS revealed that cooperative interactions between three polar residues located at the same helical face provide adequate stability for OutC self-assembly. An interhelical H-bonding mediated by Gln29 appears to be the main driving force, and two Arg residues located at the TMS boundaries are essential for the stabilization of OutC oligomers. Stepwise mutagenesis of these residues gradually diminished OutC functionality and self-association ability. The triple OutC mutant R15V/Q29L/R36A became monomeric and nonfunctional. Self-association and functionality of the triple mutant were partially restored by the introduction of a polar residue at an alternative position in the interhelical interface. Thus, the OutC TMS is more than just a membrane anchor; it drives the protein self-association that is essential for formation of a functional secretion system. The type II secretion system (T2SS) 2The abbreviations used are: T2SS, type II secretion system; BTH, bacterial two-hybrid; GSP, general secretion pathway; GST, glutathione S-transferase; PDZ, post-synaptic density, Disc large and Zo-1 proteins; TMS, transmembrane (TM) segment; Ni-NTA, nickel-nitrilotriacetic acid; Stokes radius, RS; aa, aminoacids; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is employed by a number of pathogenic Gram-negative bacteria to secrete lytic enzymes and toxins (1Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (222) Google Scholar). Secretion via this pathway is a two-step process. The proteins first cross the cytoplasmic membrane either by the Sec system or by the twin-arginine transport system, Tat (2Voulhoux R. Ball G. Ize B. Vasil M.L. Lazdunski A. Wu L.F. Filloux A. EMBO J. 2001; 20: 6735-6741Crossref PubMed Scopus (214) Google Scholar). Once exported into the periplasm, the proteins are then secreted by the T2SS across the outer membrane into the medium. Depending on the species, the secretion machinery consists of 12–15 proteins whose exact function is still obscure for most of them. The majority of the components of T2SS are highly conserved, and most of the corresponding genes can be swapped between diverse bacterial species, except for gspC and gspD (3Lindeberg M. Salmond G.P.C. Collmer A. Mol. Microbiol. 1996; 20: 175-190Crossref PubMed Scopus (96) Google Scholar, 4Possot O.M. Vignon G. Bomchil N. Ebel F. Pugsley A.P. J. Bacteriol. 2000; 182: 2142-2152Crossref PubMed Scopus (111) Google Scholar) (gsp for general secretory pathway (5Pugsley A.P. Microbiol. Rev. 1993; 57: 50-108Crossref PubMed Google Scholar)). The T2SS of the phytopathogenic enterobacteria Erwinia chrysanthemi, referred to as the Out system, secretes several pectinases and a cellulase (6Bouley J. Condemine G. Shevchik V.E. J. Mol. Biol. 2001; 308: 205-219Crossref PubMed Scopus (78) Google Scholar). Curiously, most of the proteins composing the T2SS are associated with or integrated in the inner membrane, except for OutD and OutS, which are located in the outer membrane (7Shevchik V.E. Robert-Baudouy J. Condemine G. EMBO J. 1997; 16: 3007-3016Crossref PubMed Scopus (142) Google Scholar, 8Shevchik V.E. Condemine G. Microbiology. 1998; 144: 3219-3228Crossref PubMed Scopus (63) Google Scholar). This suggests that certain components of the T2SS ensure a permanent or transient junction between the two cellular membranes to allow for a functional integrity of the secretion machinery. The existence of two separate steps in the T2S pathway assumes that the secreted proteins, once they have been exported into the periplasm, should be recognized by a special element(s) of the T2S machinery. The inner membrane protein OutC has been suggested for the roles of signal transduction between the two cell membranes and recognition of secreted proteins (3Lindeberg M. Salmond G.P.C. Collmer A. Mol. Microbiol. 1996; 20: 175-190Crossref PubMed Scopus (96) Google Scholar, 6Bouley J. Condemine G. Shevchik V.E. J. Mol. Biol. 2001; 308: 205-219Crossref PubMed Scopus (78) Google Scholar, 7Shevchik V.E. Robert-Baudouy J. Condemine G. EMBO J. 1997; 16: 3007-3016Crossref PubMed Scopus (142) Google Scholar, 9Bleves S. Gérard-Vincent M. Lazdunski A. Filloux A. J. Bacteriol. 1999; 181: 4012-4019Crossref PubMed Google Scholar). OutC consists of a short cytoplasmic sequence, a single transmembrane segment (TMS), and a large periplasmic region (10Thomas J.D. Reeves P.J. Salmond G.P.C. Microbiology. 1997; 143: 713-720Crossref PubMed Scopus (49) Google Scholar). A putative PDZ domain is located close to its C terminus (11Pallen M.J. Ponting C.P. Mol. Microbiol. 1997; 26: 411-415Crossref PubMed Scopus (27) Google Scholar). GspC proteins from certain other bacteria presumably possess a coiled-coil domain instead of a PDZ domain (12Peabody C.R. Chung Y.J. Yen M.R. Vidal-Ingigliardi D. Pugsley A.P. Saier Jr., M.H. Microbiology. 2003; 149: 3051-3072Crossref PubMed Scopus (284) Google Scholar). Some algorithms also predict a coiled-coil structure for Erwinia OutC. Regardless of its structure, inter-species swapping indicated that this region of OutC directly participates in the specific recognition of the secreted proteins (6Bouley J. Condemine G. Shevchik V.E. J. Mol. Biol. 2001; 308: 205-219Crossref PubMed Scopus (78) Google Scholar). Recently it was proposed that this region of GspC could be involved in the formation of homo-multimeric complexes (13Gérard-Vincent M. Robert V. Ball G. Bleves S. Michel G.P. Lazdunski A. Filloux A. Mol. Microbiol. 2002; 44: 1651-1665Crossref PubMed Scopus (43) Google Scholar). Genetic and biochemical studies suggested that GspC could interact with the inner membrane proteins GspM and GspL (14Lee H.M. Chen J.R. Lee H.L. Leu W.M. Chen L.Y. Hu N.T. J. Bacteriol. 2004; 186: 2946-2955Crossref PubMed Scopus (21) Google Scholar, 15Possot O.M. Gérard-Vincent M. Pugsley A.P. J. Bacteriol. 1999; 181: 4004-4011Crossref PubMed Google Scholar, 16Robert V. Hayes F. Lazdunski A. Michel G.P. J. Bacteriol. 2002; 184: 1779-1782Crossref PubMed Scopus (22) Google Scholar). Furthermore, the current models of the T2SS imply that GspC interacts, at least transiently, with the outer membrane protein GspD (1Filloux A. Biochim. Biophys. Acta. 2004; 1694: 163-179Crossref PubMed Scopus (222) Google Scholar, 7Shevchik V.E. Robert-Baudouy J. Condemine G. EMBO J. 1997; 16: 3007-3016Crossref PubMed Scopus (142) Google Scholar, 9Bleves S. Gérard-Vincent M. Lazdunski A. Filloux A. J. Bacteriol. 1999; 181: 4012-4019Crossref PubMed Google Scholar, 15Possot O.M. Gérard-Vincent M. Pugsley A.P. J. Bacteriol. 1999; 181: 4004-4011Crossref PubMed Google Scholar, 17Lee H.M. Wang K.C. Liu Y.L. Yew H.Y. Chen L.Y. Leu W.M. Chen D.C. Hu N.T. J. Bacteriol. 2000; 182: 1549-1557Crossref PubMed Scopus (33) Google Scholar). It has been shown that some components of the T2SS are assembled into homomultimeric structures. The NTPase GspE located in the cytoplasm seems to take the shape of a hexameric ring-like structure (18Robien M.A. Krumm B.E. Sandkvist M. Hol W.G.J. J. Mol. Biol. 2003; 333: 657-674Crossref PubMed Scopus (104) Google Scholar). When overexpressed, certain pseudopilins form long flexible pili comprising multiple pseudopilin subunits (19Sauvonnet N. Vignon G. Pugsley A.P. Gounon P. EMBO J. 2000; 19: 2221-2228Crossref PubMed Scopus (188) Google Scholar). The secretin GspD forms dodecameric rings in a lipid bilayer that could correspond to the channels in the bacterial outer membranes (20Bitter W. Koster M. Latijnhouwers M. de Cock H. Tommassen J. Mol. Microbiol. 1998; 27: 209-219Crossref PubMed Scopus (194) Google Scholar, 21Nouwen N. Stahlberg H. Pugsley A.P. Engel A. EMBO J. 2000; 19: 2229-2236Crossref PubMed Scopus (108) Google Scholar). Therefore, it seems plausible that OutC, which was presumed to interact with OutD and with the inner membrane platform formed by OutE, OutF, OutL, and OutM (22Py B. Loiseau L. Barras F. EMBO Rep. 2001; 2: 244-248Crossref PubMed Scopus (129) Google Scholar), could also be assembled into multimeric structures. The mechanisms that govern the assembly of the T2SS components into a functional multiprotein complex are still poorly understood. Certain binary interactions between soluble protein regions have been detected by using yeast two-hybrid analysis and in vitro assays (22Py B. Loiseau L. Barras F. EMBO Rep. 2001; 2: 244-248Crossref PubMed Scopus (129) Google Scholar, 23Sandkvist M. Keith J.M. Bagdasarian M. Howard S.P. J. Bacteriol. 2000; 182: 742-748Crossref PubMed Scopus (49) Google Scholar). Although specific interactions between α-helical TMS are important for the folding and oligomerization of membrane proteins (24Gurran A.R. Engelman D.M. Curr. Opin. Struct. Biol. 2003; 13: 412-417Crossref PubMed Scopus (200) Google Scholar), their role in the assemblage and function of the T2SS has not been thoroughly analyzed. Here we performed a detailed analysis of the oligomerization state of OutC. Bacterial two-hybrid, cross-linking and pull-down assays revealed that the self-association of OutC is driven by the TMS, whereas the periplasmic region is dispensable for self-association. Site-directed mutagenesis of the TMS revealed that cooperative interactions between three polar residues located at the same helical face, Gln29, Arg15, and Arg36, provide adequate stability for the OutC self-assembly necessary for the protein function. These results allowed us to revise the previous opinion that a single TMS of GspC plays a passive role assuming the anchoring of the protein into the inner membrane (15Possot O.M. Gérard-Vincent M. Pugsley A.P. J. Bacteriol. 1999; 181: 4004-4011Crossref PubMed Google Scholar), and we demonstrated instead that the TMS drives the self-association of OutC that is essential for the formation of a functional secretion system. Plasmids—The plasmids used in the study are listed in Table 1. The single and multiple mutations were introduced in the outC sequence of E. chrysanthemi 3937 by site-directed mutagenesis using the QuikChange kit (Stratagene). The primers used are listed in supplemental Table S1. The nucleotide sequences of mutant genes were systematically checked (Genome Express). The OutC truncated derivatives were constructed by using the restriction sites introduced by site-directed mutagenesis and naturally existing sites to give in frame deletions, as indicated in Table 1.TABLE 1Plasmids used in this studyPlasmidGenotype/phenotypeReferenceTwo-hybrid vectorspUT18CpUC19 derivative coding T18 fragment of CyaA upstream of a multicloning site, ApR25Karimova G. Pidoux J. Ullmann A. Ladant D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5752-5756Crossref PubMed Scopus (1182) Google ScholarpKT25pSU40 derivative coding T25 fragment of CyaA upstream of a multicloning site, KnR25Karimova G. Pidoux J. Ullmann A. Ladant D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5752-5756Crossref PubMed Scopus (1182) Google ScholarpUT-oCpUT18C carrying outC (aa 2-272) fused in-frame to the T18 geneThis workpUT-oCΔApUT-oC with a BsaHI-BsaHI deletion (Δ aa 100-123)This workpUT-oCΔRpUT-oC with a HindII-BsrBI deletion (Δ aa 173-256)This workpUT-oCΔHpUT-oC with a HpaIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis.-NruIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis. deletion (Δ aa 61-72)This workpUT-oCΔNpUT-oC with a NaeI-HindII deletion (Δ aa 103-172)This workpUT-oCΔUpUT-oC with a SmaIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis.-NruIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis. deletion (D aa 41-72)This workpUT-oCΔLpUT18C carrying truncated outC fused at BamHIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis. (aa 161-272)This workpUT-oCΔSpUT18C carrying truncated outC fused at SmaIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis. (aa 40-272)This workpUT-oC-TMSpUT-oC with Opal stop codon at 43 aa (Δ aa 43-272)This workpKT-oCpKT25 carrying outC (aa 2-272) fused in-frame to the T25 geneThis workpKT-oCΔRpKT-oC with a HindII-BsrBI deletion (Δ aa 173-256)This workpKT-oCΔNpKT-oC with a NaeI-HindII deletion (Δ aa 103-172)This workpKT-oCΔUpKT-oC with a SmaIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis.-NruIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis. deletion (Δ aa 41-72)This workpKT-oCΔCpKT25 carrying truncated outC fused at SpeIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis. (aa 14-272)This workpKT-oCΔLpKT25 carrying truncated outC fused at BamHIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis. (aa 161-272)This workpKT-oCΔSpKT25 carrying truncated outC fused at SmaIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis. (aa 40-272)This workpKT-oC-TMSpKT25 with Opal stop codon at 43 aa (Δ aa 43-272)This workGST fusion vectorsPGEX-6P-3GST fusion vector with PreScission protease cleavage site, ApRGE HealthcarepGX-oCpGEX-6P-3 carrying outC (aa 2-272) fused in-frame to the GST geneThis workpGX-oCΔApGX-oC with a BsaHI-BsaHI deletion (Δ aa 100-123)This workpGX-oCΔHpGX-oC with a HpaIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis.-NruIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis. deletion (Δ aa 61-72)This workpGX-oCΔNpGX-oC with a NaeI-HindII deletion (Δ aa 103-172)This workpGX-oCΔRpGX-oC with a HindII-BsrBI deletion (Δ aa 173-256)This workpGX-oCΔUpGX-oC with a SmaIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis.-NruIaRestriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis. deletion (Δ aa 41-72)This workpGX-oCΔSpGEX-6P-3 carrying truncated outC (aa 40-272) fused in-frame to the GST geneThis workpGX-oC-TMSpGX-oC with Opal stop codon at 43 aa (Δ aa 43-272)This workHis6 fusion vectorspQE-30His6 fusion vectors, ApRQiagenpQE-32pQE-HisOCpQE-32 carrying His6-outC (aa 2-272)6Bouley J. Condemine G. Shevchik V.E. J. Mol. Biol. 2001; 308: 205-219Crossref PubMed Scopus (78) Google ScholarpQE-HisOCΔLpQE-30 carrying His6-outC (aa 161-272)This workpQE-HisOCΔSpQE-30 carrying His6-outC (aa 40-272)This workpTdB-OCpT7-6 carring outC under the pelC promoter6Bouley J. Condemine G. Shevchik V.E. J. Mol. Biol. 2001; 308: 205-219Crossref PubMed Scopus (78) Google Scholara Restriction endonuclease sites introduced in the outC sequence by site-directed mutagenesis. Open table in a new tab Bacterial Two-hybrid Experiments—The bacterial two-hybrid system, kindly provided by G. Karimova (25Karimova G. Pidoux J. Ullmann A. Ladant D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5752-5756Crossref PubMed Scopus (1182) Google Scholar), was used according to the authors' instructions. outC or its truncated derivatives were fused in phase to the C termini of gene fragments coding for the T18 and T25 domains of adenylate cyclase on the plasmids pUT18C and pKT25, respectively (Table 1 and Fig. 1A). Various combinations of constructs were co-transformed into the cya Escherichia coli strain DHP1 (F– cya glnV44(AS) recA1 endA1 gyrA96 (Nalr) thi1 hsdR17 spoT1 rfbD1) (25Karimova G. Pidoux J. Ullmann A. Ladant D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5752-5756Crossref PubMed Scopus (1182) Google Scholar) and the transformants were plated on MacConkeymaltose agar supplemented with ampicillin and kanamycin. The color of the colonies was monitored during incubation at 30 °C for 36–48 h. β-Galactosidase assays were performed as described (26Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972: 352-355Google Scholar) in DHP1 liquid cultures grown in Luria-Bertani (LB) medium supplemented with 1 mm isopropyl-β-d-thiogalactopyranoside and with antibiotics at 28 °C for 18 h. All assays were performed from triplicate cultures on three to four different bacterial transformants and on several different days. Gel Electrophoresis and Immunoblotting—SDS-PAGE was performed according to Laemmli (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), and proteins were either stained with Coomassie G-250 or transferred onto nitrocellulose. The membrane was then incubated with antibodies and developed with the ECL detection kit (GE Healthcare) as described previously (7Shevchik V.E. Robert-Baudouy J. Condemine G. EMBO J. 1997; 16: 3007-3016Crossref PubMed Scopus (142) Google Scholar). The primary antibodies used were 1:10,000-diluted anti-BlaM (Chemicon), 1:4000-diluted anti-Cya (provided by G. Karimova), anti-OutC, anti-PelD, anti-PemA, and anti-Cel5 as described previously (6Bouley J. Condemine G. Shevchik V.E. J. Mol. Biol. 2001; 308: 205-219Crossref PubMed Scopus (78) Google Scholar). Horseradish peroxidase-conjugated Ni-NTA (Qiagen) was diluted 1:3000. Complementation Test—To test the functionality of OutC mutant proteins, the E. chrysanthemi ΔoutC strain A3618 (6Bouley J. Condemine G. Shevchik V.E. J. Mol. Biol. 2001; 308: 205-219Crossref PubMed Scopus (78) Google Scholar) was transformed with a pTdB-OC derivative carrying a corresponding outC mutant gene. Exoprotein secretion was initially tested using the halo size on plate assays for pectinase and cellulase activities (6Bouley J. Condemine G. Shevchik V.E. J. Mol. Biol. 2001; 308: 205-219Crossref PubMed Scopus (78) Google Scholar). For immunoblotting assays, E. chrysanthemi were grown at 28 °C in LB for 14 h until early stationary phase. Cells were pelleted by centrifugation at 10,000 × g for 2 min and resuspended at the same volume of LB. The culture supernatants and cell extracts were separated by SDS-PAGE and revealed with antibodies against diverse exoproteins. Protease Accessibility Assay—E. coli MG1655 (F′ λ– rph-1) cells carrying a plasmid with one of the outC derivatives were grown in LB at 30 °C to an A600 of 0.6. Cells from 5 ml of cultures were pelleted and resuspended in 0.2 ml of 0.1 m Tris-HCl (pH 8.0), 0.5 m sucrose, and 1 mm EDTA. Lysozyme (5 μl of 3 mg/ml) was added, and the cells were incubated on ice for 15 min. After the incubation, 0.2 ml of ice-cold 5 mm MgSO4 was added, and the spheroplast suspension was separated into 100-μl aliquots. Two aliquots were treated with trypsin (50 μg/ml) for 15 min on ice and, before the proteolysis, 0.05% Triton X-100 was added to one of them. Proteolysis was stopped by the addition of phenylmethylsulfonyl fluoride to 2 mm, and the spheroplasts were harvested at 4000 × g for 4 min and resuspended in the same volume of Laemmli sample buffer. An untreated spheroplast aliquot was used for β-galactosidase and alkaline phosphatase activity assays (26Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972: 352-355Google Scholar). Protein Purification—E. coli NM522 (New England Biolabs) cells carrying a plasmid coding for one of the His-tagged OutC proteins (Table 1) were grown in LB supplemented with ampicillin (150 μg/ml) at 30 °C. At an A600 of 0.7, isopropyl-β-d-thiogalactopyranoside was added to 1 mm, and the cultures were grown for an additional 3 h. Cells were pelleted by centrifugation and frozen at –70 °C. The cell pellet was resuspended in 100 mm sodium phosphate, 10 mm Tris-HCl, 100 mm NaCl, 1% Triton X-100 (pH 8.0) (buffer A), and broken by sonication. The recombinant proteins were purified on Ni-NTA agarose (Qiagen) at 15 °C as described by the manufacturer. Pefabloc (0.1 mg/ml) was used in all solutions. E. coli BL21(DE3) (Stratagene) cells carrying a plasmid coding for one of the GST-OutC derivatives were grown and stored as above. Purification was performed at 15 °C. The cell pellet was resuspended in 50 mm Tris-HCl, 100 mm NaCl, 1 mm EDTA, 1% Triton X-100 (pH 7.0) (buffer B), and sonicated. The lysate was centrifuged at 7000 × g for 5 min, mixed with glutathione-Sepharose 4B (GE Healthcare) equilibrated in the same buffer, and then incubated with mixing for 1 h. Unbound proteins were removed by washing 3 times for 5 min with buffer B and an additional 3 times with buffer B containing 0.1% Triton X-100. The proteins were cleaved from GST by the addition of PreScission protease (GE Healthcare) to 35 units/ml for 2 h. Eluted proteins were separated from the resin by centrifugation at 1000 × g for 2 min and mixed with a new portion of the resin to eliminate any trace of uncleaved GST fusions and the PreScission protease. Pulldown Assay—GST-OutC derivatives were purified as above except that the PreScission protease was omitted, and the fusion proteins remained immobilized on glutathione-Sepharose beads. The quantities of immobilized GST fusions were checked by SDS-PAGE before the binding assays. An equal amount (about 50 μg) of purified His-tagged OutC or 0.6 ml of BL21 lysate containing OutC mutant proteins were added to immobilized GST or GST-OutC fusions in buffer B containing 0.1% Triton X-100. After a 1-h incubation with mixing at 8 rpm and at 15 °C, the mixtures were spun for 2 min at 1000 × g, and the pelleted beads were washed 3 times with the same buffer. The bound proteins were eluted with Laemmli sample buffer and analyzed by SDS-PAGE and immunoblotting with Ni-NTA-peroxidase or anti-OutC. Gel Filtration Chromatography—A Superdex 200 10/300 GL column (GE Healthcare) was equilibrated with buffer B containing 0.1% Triton X-100 at 15 °C. The flow rate was 0.4 ml/min, and 0.2-ml fractions were collected. The fractions were analyzed by SDS-PAGE followed by immunoblotting with anti-OutC. Blue dextran and NaCl were used for the determination of the void volume (Vo) and the total volume (Vt), respectively. The elution volumes (Ve) of proteins of known molecular mass and Stokes radius (RS) were used as the standards. The standard curve was plotted with the logarithm of RS against the KD of the standard protein (KD = Ve – Vo/Vt – Vo). Determination of Sedimentation Coefficient—Linear 3.8-ml sucrose gradients of 2.5–20% sucrose (w/v) in buffer B containing 0.1% Triton X-100 were prepared in either H2O or D2O. The sample of 60 μl, containing OutC or one of its derivatives, together with the standard proteins was loaded onto the gradients and centrifuged for 14 h (H2O) or 20 h (D2O) at 55,000 rpm in a Beckman SW-60 rotor at 4 °C. Then forty fractions were collected from the bottom of each gradient and analyzed by SDS-PAGE. The positions of the proteins were determined by quantitative scanning of stained gels and immunoblots. Refractive indices were determined with a Carl Zeiss refractometer. The partial specific volumes (ν) of proteins were calculated from the amino acid composition using Sednterp software. The ν of Triton X-100 was considered to be 0.908 ml/g (28Tanford C. Nozaki Y. Reynolds J.A. Makino S. Biochemistry. 1974; 13: 2369-2376Crossref PubMed Scopus (378) Google Scholar), whereas ν of the protein-detergent complex was determined from data obtained by H2O and D2O gradient sedimentation as described (28Tanford C. Nozaki Y. Reynolds J.A. Makino S. Biochemistry. 1974; 13: 2369-2376Crossref PubMed Scopus (378) Google Scholar). The apparent molecular mass, the frictional ratio (f/f0) of the protein-detergent complex, and the amount of detergent bound to the protein were calculated as described (29Siegel L.M. Monty K.J. Biochim. Biophys. Acta. 1966; 112: 346-362Crossref PubMed Scopus (1547) Google Scholar, 30Sadler J.E. J. Biol. Chem. 1979; 254: 4443Abstract Full Text PDF PubMed Google Scholar). The axial ratio (a/b) was estimated from f/f0 using the Perrin's function (P) as described (31Harding S.E. Cölfen H. Anal. Biochem. 1995; 228: 131-142Crossref PubMed Scopus (49) Google Scholar), with the apparent hydration (δ) values calculated using Sednterp. Bacterial Two-hybrid Assays Detect OutC Homodimers—To determine whether OutC is able to self-interact in vivo, we used the bacterial two-hybrid (BTH) system (25Karimova G. Pidoux J. Ullmann A. Ladant D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5752-5756Crossref PubMed Scopus (1182) Google Scholar). In BTH the physical association of the two interacting proteins is spatially separated from the transcriptional events (via cAMP synthesis) so it is possible to analyze protein interactions that occur either in the bacterial cytoplasm or in the inner membrane. Full-length OutC fused to the C termini of T18 and T25 fragments were protease-sensitive in a spheroplast assay (Fig. 2 and not shown), indicating that the OutC moiety takes a correct Nin, Cout topology in the inner membrane, as does intact OutC. The E. coli DHP1 cells expressing these two fusions formed red colonies on MacConkey plates, whereas the negative controls (one of the fusions combined with an empty vector) appeared white (Table 2). A positive control (pUT18-zip and pKT25-zip) formed dark red colonies. The level of β-galactosidase activity measured with the two OutC fusions was 5–7-fold higher than that of the negative control and was comparable with the positive control (Table 2). This clearly indicates OutC self-interaction in vivo. Because the OutC moiety in the fusion acquired the correct membrane topology, the OutC homodimerization detected by BTH could be results of interactions in the cytoplasmic membrane, the periplasm, or both.TABLE 2OutC self-interaction detected by the bacterial two-hybrid systemFused proteinPhenotype on MacConkey maltoseβ-Galactosidase activityT25T18units/ml ± S.D.White5,800 ± 500Leucine-ZipLeucine-ZipDark red46,500 ± 5,400OutCWhite5,700 ± 400OutCWhite6,900 ± 600OutCOutCRed38,000 ± 4,000OutCΔCΔ1-13OutCRed37,000 ± 3,300OutCΔRΔ173-256OutCΔRΔ173-256Red23,100 ± 2,700OutCΔHΔ61-72OutCRed35,300 ± 3,800OutCΔNΔ103-172OutCΔAΔ100-123Red36,000 ± 3,000OutCΔNΔ103-172OutCΔNΔ103-172Red40,500 ± 3,800OutCΔUΔ41-72OutCΔUΔ41-72Red22,800 ± 3,300OutC-TMSOutC-TMSRed35,400 ± 3,600OutCΔSΔ1-39OutCΔSΔ1-39White/center red7,500 ± 1,200OutCΔLΔ1-160OutCΔLΔ1-160White/center red7,800 ± 1,000 Open table in a new tab The TMS Is Essential for the OutC Homodimerization in BTH—To define the regions involved in homodimerization, a series of OutC deletion mutants fused to the C termini of T18 and T25 fragments was constructed (Fig. 1A and Tables 1 and 2). Immunoblotting tests with anti-OutC and anti-Cya were systematically performed throughout the BTH assays to check the amounts of truncated hybrids (not shown). When the short cytoplasmic region was deleted (OutCΔC), the β-galactosidase activity was equivalent to that with the full-length fusions (Table 2), indicating that this region is not involved in OutC dimerization. The fusions carrying deletions in the periplasmic region (OutCΔA, OutCΔH, OutCΔN, OutCΔR, and OutCΔU) were sensitive to trypsin in a spheroplast assay, and thus, they were exposed in the periplasm as is full-length OutC (Fig. 2 and not shown). When these truncated fusions were co-expressed pair-wise, red colonies were formed on MacConkey plates (Table 2