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• W2022132556 abstract "The binding protein-dependent maltose transport system of enterobacteria (MalFGK2), a member of the ATP-binding cassette (ABC) transporter superfamily, is composed of two integral membrane proteins, MalF and MalG, and of two copies of an ATPase subunit, MalK, which hydrolyze ATP, thus energizing the translocation process. In addition, an extracellular (periplasmic) substrate-binding protein (MalE) is required for activity. Ligand translocation and ATP hydrolysis are dependent on a signaling mechanism originating from the binding protein and traveling through MalF/MalG. Thus, subunit-subunit interactions in the complex are crucial to the transport process but the chemical nature of residues involved is poorly understood. We have investigated the proximity of residues in a conserved sequence (“EAA” loop) of MalF and MalG to residues in a helical segment of the MalK subunits by means of site-directed chemical cross-linking. To this end, single cysteine residues were introduced into each subunit at several positions and the respectivemalF and malG alleles were individually co-expressed with each of the malK alleles. Membrane vesicles were prepared from those double mutants that contained a functional transporter in vivo and treated with Cu(1,10-phenanthroline)2SO4 or bifunctional cross-linkers. The results suggest that residues Ala-85, Lys-106, Val-114, and Val-117 in the helical segment of MalK, to different extents, participate in constitution of asymmetric interaction sites with the EAA loops of MalF and MalG. Furthermore, both MalK monomers in the complex are in close contact to each other through Ala-85 and Lys-106. These interactions are strongly modulated by MgATP, indicating a structural rearrangement of the subunits during the transport cycle. These data are discussed with respect to current transport models. The binding protein-dependent maltose transport system of enterobacteria (MalFGK2), a member of the ATP-binding cassette (ABC) transporter superfamily, is composed of two integral membrane proteins, MalF and MalG, and of two copies of an ATPase subunit, MalK, which hydrolyze ATP, thus energizing the translocation process. In addition, an extracellular (periplasmic) substrate-binding protein (MalE) is required for activity. Ligand translocation and ATP hydrolysis are dependent on a signaling mechanism originating from the binding protein and traveling through MalF/MalG. Thus, subunit-subunit interactions in the complex are crucial to the transport process but the chemical nature of residues involved is poorly understood. We have investigated the proximity of residues in a conserved sequence (“EAA” loop) of MalF and MalG to residues in a helical segment of the MalK subunits by means of site-directed chemical cross-linking. To this end, single cysteine residues were introduced into each subunit at several positions and the respectivemalF and malG alleles were individually co-expressed with each of the malK alleles. Membrane vesicles were prepared from those double mutants that contained a functional transporter in vivo and treated with Cu(1,10-phenanthroline)2SO4 or bifunctional cross-linkers. The results suggest that residues Ala-85, Lys-106, Val-114, and Val-117 in the helical segment of MalK, to different extents, participate in constitution of asymmetric interaction sites with the EAA loops of MalF and MalG. Furthermore, both MalK monomers in the complex are in close contact to each other through Ala-85 and Lys-106. These interactions are strongly modulated by MgATP, indicating a structural rearrangement of the subunits during the transport cycle. These data are discussed with respect to current transport models. ATP-binding cassette transporter membrane-bound maltose transport complex containing the hydrophobic subunits MalF and MalG, and the ATPase subunit MalK Cu(1,10-phenanthroline)2SO4 dithiothreitol 1,6-bismaleimidohexane N,N′-o-phenylenedimaleimide N,N′-p-phenylenedimaleimide polyacrylamide gel electrophoresis The rapidly growing family of ATP-binding cassette (ABC)1 transport systems comprises an extremely diverse class of membrane transport proteins that couple the energy of ATP hydrolysis to the translocation of solutes across biological membranes (1.Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3374) Google Scholar). Prominent members of the family include the P-glycoprotein involved in multidrug resistance of certain cancer cells; the cystic fibrosis transmembrane regulator protein, which is mutated in patients affected by cystic fibrosis; the TAP1-TAP2 peptide transporter, associated with antigen presentation (1.Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3374) Google Scholar); a transporter that, when mutated, causes adrenoleukodystrophy, an error in peroxisomal β-oxidation of long fatty acids (2.Mosser J. Douar A.-M. Sarde C.-O. Kioschis P. Feil R. Moser H. Poustka A.M. Mandel J.L. Aubourg P. Nature. 1993; 361: 726-730Crossref PubMed Scopus (1000) Google Scholar); a protein involved in recessive Stargardt's macula dystrophy (3.Allikmets R. Singh N. Sun H. Shroyer N.F. Hutchinson A. Chidambaram A. Gerrard B. Baird L. Stauffer D. Peiffer A. Rattner A. Smallwood P. Li Y. Anderson K.L. Lewis R.A. Nathans J. Leppert M. Dean M. Lupski J.R. Nat. Genet. 1997; 15: 236-246Crossref PubMed Scopus (1102) Google Scholar); and the subclass of binding protein-dependent ABC transport systems that mediate the uptake of a large variety of nutrients in prokaryotes (4.Boos W. Lucht J.M. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low Jr., K.B. Magasanik B. Rezinkoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, DC1996: 1175-1209Google Scholar). A prototype ABC transporter is composed of four entities: two membrane-integral domains, which presumably constitute a translocation pore; and two domains carrying the highly conserved nucleotide binding motifs (“Walker” sites A and B) (also referred to as ABC subunits/domains), which are thought to provide the energy for the transport process (1.Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3374) Google Scholar). The ABC domains are further characterized by a unique “signature sequence” (LSGGQ motif) of yet unknown function located at the C-terminal end of a large helical peptide segment that connects the Walker A and B sites (5.Schneider E. Hunke S. FEMS Microbiol. Rev. 1998; 22: 1-20Crossref PubMed Google Scholar). In export systems, these modules are mostly fused to yield a single polypeptide chain, while prokaryotic ABC transporters mediating the uptake of solutes are built up from individual subunits (1.Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3374) Google Scholar). The latter also require an additional extracellular (periplasmic) substrate-binding protein for activity (4.Boos W. Lucht J.M. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low Jr., K.B. Magasanik B. Rezinkoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, DC1996: 1175-1209Google Scholar). The binding protein-dependent maltose transporter of enterobacteria, such as Escherichia coli andSalmonella typhimurium, is a well characterized model system for studying the mechanism of action of the ABC transport family. It is composed of a soluble periplasmic receptor, the maltose-binding protein, MalE, and of a membrane-bound complex (MalFGK2), consisting of one copy each of the hydrophobic subunits MalF and MalG and of two copies of the nucleotide-binding subunit MalK (6.Boos W. Shuman H. Microbiol. Mol. Biol. Rev. 1998; 62: 204-229Crossref PubMed Google Scholar). Purified MalK displays a spontaneous ATPase activity (7.Morbach S. Tebbe S. Schneider E. J. Biol. Chem. 1993; 268: 18617-18621Abstract Full Text PDF PubMed Google Scholar), while, in the MalFGK2 complex, ATP hydrolysis and ligand translocation are strictly coupled and dependent on the binding protein (8.Davidson A.L. Nikaido H. J. Biol. Chem. 1991; 266: 8946-8951Abstract Full Text PDF PubMed Google Scholar). The presence of substrate in the medium is thought to be signaled by liganded MalE via interaction with externally exposed peptide loops of MalF and MalG (9.Davidson A.L. Shuman H.A. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2360-2364Crossref PubMed Scopus (236) Google Scholar). As a consequence, conformational changes of the latter are transmitted to the MalK subunits, which, in turn, become activated. Hydrolysis of ATP would then trigger subsequent conformational changes that eventually lead to the translocation of the substrate molecule. Obviously, specific interactions between the individual subunits of the transport complex are crucial to this model. The chemical nature of residues involved in such an interplay between the hydrophobic components and the ATPase subunits is largely unknown. However, recent genetic and biochemical studies provided first, albeit indirect, evidence in favor of amino acids located in a mostly helical segment of MalK (approximately encompassing residues 83–149) (see Fig.1 A) as putative candidates for contacting MalF and/or MalG. By using a close homologue, the LacK protein of Agrobacterium radiobacter, as a tool, Wilken et al. (10.Wilken S. Schmees G. Schneider E. Mol. Microbiol. 1996; 22: 555-666Crossref PubMed Scopus (112) Google Scholar) demonstrated that mutations affecting Val-114 (→ Met) and Leu-123 (→ Phe) optimize the capability of LacK to substitute for MalK in maltose transport. Furthermore, reconstitution experiments revealed that Lys-106 of MalK, which is susceptible to trypsin in the purified protein (11.Schneider E. Wilken S. Schmid R. J. Biol. Chem. 1994; 269: 20456-20461Abstract Full Text PDF PubMed Google Scholar), is protected against proteolytic attack in the assembled complex (12.Mourez M. Jéhano M. Schneider E. Dassa E. Mol. Microbiol. 1998; 30: 353-363Crossref PubMed Scopus (33) Google Scholar). Moreover, and most importantly, Mourez et al. (13.Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar) identified substitutions of methionine for Ala-85, Val-117, or Val-149 of MalK, as suppressors of mutations affecting a conserved sequence motif in the membrane-integral components MalF and MalG. This motif (consensus: EAA … G. . . . . . . . . . .I-LP, thus often referred to as “EAA loop”) (see Fig. 1 B) is located in a cytoplasmic loop at a distance of ∼100 residues from the C terminus (14.Dassa E. Hofnung M. EMBO J. 1985; 4: 2287-2293Crossref PubMed Scopus (144) Google Scholar). It is shared by the hydrophobic subunits of prokaryotic binding protein-dependent ABC transporters (4.Boos W. Lucht J.M. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low Jr., K.B. Magasanik B. Rezinkoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, DC1996: 1175-1209Google Scholar), but a similar motif has recently been identified in eukaryotic members of the family (15.Shani N. Sapag A. Valle D. J. Biol. Chem. 1996; 271: 8725-8730Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In this paper, we provide the first direct evidence for interaction of the helical domain of an ABC subunit and the EAA loops of the membrane-integral components that is modulated by ATP. The neighboring residues are revealed by the introduction of single cysteines into each subunit and disulfide bridge formation or chemical cross-linking between MalK monomers, MalK and MalF, and MalK and MalG, respectively. E. coli strain ED169 (F− ΔlacU169 araD139 rpsL relA thi flbBΔmalB107) is described elsewhere (13.Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar). Plasmids used in this study are listed in Table I.Table IPlasmids used in this studyPlasmidRelevant genotypeSource or referencepSE380trcpromoterStratagenepSU19PA15 ori17.Martinez E. Bartolomé B. de la Cruz F. Gene (Amst .). 1988; 68: 159-162Crossref PubMed Scopus (253) Google ScholarpTZ18Rtac promoterPharmaciapSH30malK798 (C40S, C350S, C360S) on pSE38016pSH34malK813 (C40S, C350S, C360S, V114C) on pSE380This studypSH36malK815 (C40S, C350S, C360S, A85C) on pSE380This studypSH37malK816(C40S, C350S, C360S, V117C) on pSE380This studypSH38malK817 (C40S, C350S, C360S, K106C) on pSE380This studypSH46malK798 (C40S, C350S, C360S) on pSU19This studypSH47malK813 (C40S, C350S, C360S, V114C) on pSU19This studypSH49malK815 (C40S, C350S, C360S, A85C) on pSU19This studypSH50malK816 (C40S, C350S, C360S, V117C) on pSU19This studypSH51malK817(C40S, C350S, C360S, K106C) on pSU19This studypTZFGQmalF malG on pTZ18R13.Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google ScholarpTAZFGQ*malF (Cys−)/malG (Cys−) on pTZ18RThis studypTAZFGQ(E1C/−)malF(E401C)/malG (Cys−) on pTZ18RThis studypTAZFGQ(S3C/−)malF (S403C)/malG (Cys−) on pTZ18RThis studypTAZFGQ(−/E1C)malF(Cys−)/malG (E190C) on pTZ18RThis studypTAZFGQ(−/A3C)malF (Cys−)/malG (A192C) on pTZ18RThis studypTAZFGQ(−/G7C)malF(Cys−)/malG (G196C) on pTZ18RThis studypTLCMEmalE +13.Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar Open table in a new tab The cysteine residues of the MalK protein from S. typhimurium(Cys-40, Cys-350, Cys-360) were consecutively replaced by serines as described elsewhere (16.Hunke S. Schneider E. FEBS Lett. 1999; 448: 131-134Crossref PubMed Scopus (11) Google Scholar). The plasmid carrying the resultingmalK798 allele is a derivative of pSE380 (Apr, ptrc) and was designated pSH30 (16.Hunke S. Schneider E. FEBS Lett. 1999; 448: 131-134Crossref PubMed Scopus (11) Google Scholar). For complementation analysis, the malK798 allele was cloned as aPvuII fragment encompassing the ptrc promoter into the SmaI site of pSU19 (Cmr, P15A replicon) (17.Martinez E. Bartolomé B. de la Cruz F. Gene (Amst .). 1988; 68: 159-162Crossref PubMed Scopus (253) Google Scholar), yielding plasmid pSH46. Naturally occurring cysteine residues in the MalF (Cys-85, Cys-304, Cys-388) and MalG (Cys-106) proteins fromE. coli were substituted for by serines by subjecting themalF and malG wild type alleles on plasmid pTAZFGQ (pMB1 replicon) (13.Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar) to site-directed mutagenesis as above. The resulting plasmid was named pTAZFGQ*. Mono-Cys variants of MalK were constructed by site-directed mutagenesis using themalK798 allele (Cys-less MalK) (pSH30) as template. Base changes were introduced in codons 85 (Ala → Cys), 106 (Lys → Cys), 114 (Val → Cys), or 117 (Val → Cys), yielding plasmids pSH36 (malK815), pSH38 (malK817), pSH34 (malK813), and pSH37 (malK816), respectively. For complementation studies, the malK alleles were subcloned in pSU19 as above, yielding plasmids pSH49 (malK815), pSH51 (malK817), pSH47 (malK813), and pSH50 (malK816), respectively. Mono-Cys variants of MalF or MalG were constructed by the same protocol, usingmalF(Cys −)/malG(Cys −) (pTAZFGQ*) as template. Base changes in malF were introduced in codons 401 (Glu → Cys) and 403 (Ser → Cys), resulting in plasmids pTAZFGQ(E1C/−) and pTAZFGQ(S3C/−), respectively. Base changes in malG were introduced in codons 190 (Glu → Cys), 192 (Ala → Cys), or 196 (Glu → Cys), resulting in plasmids pTAZFGQ(−/E1C), pTAZFGQ(−/A3C), and pTAZFGQ(−/G7C), respectively. The presence of the mutations was verified by nucleotide sequence analysis. Strain ED169 (ΔmalB107) was transformed with any combination of plasmids encoding Cys-less and mono-Cys variants of MalK (derivatives of pSU19), Cys-less and mono-Cys variants of MalF or MalG (derivatives of pTZFGQ), and plasmid pTLCME (Knr, pSC101 replicon), encoding E. coli maltose-binding protein (MalE) (13.Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar). Transformants were analyzed for utilization of maltose on MacConkey medium containing 2% maltose and supplemented with ampicillin (100 μg/ml), chloramphenicol (20 μg/ml), and kanamycin (50 μg/ml). Microtiter plates were incubated at 30 °C and scored for growth after 24–48 h. Cells of strain ED169 harboring the described plasmids were grown in LB medium (18.Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar) supplemented with ampicillin, chloramphenicol, and kanamycin at 30 °C to an OD650 = 0.3. Plasmid-encoded mal gene expression was induced by the addition of 0.5 or 1 mmisopropyl-β-thiogalactoside, and growth was continued for 2 h. Cells were harvested, washed twice in M63 salt medium (18.Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar), and adjusted to to an optical density at 650 nm of 1.2. Transport assays were essentially performed as described previously (19.Schneider E. Walter C. Mol. Microbiol. 1991; 5: 1375-1383Crossref PubMed Scopus (29) Google Scholar), with the following modifications; 20 μl of [14C]maltose (44 kBq) was added to 980 μl of cells to a final concentration of 0.5 mm, and 0.3-ml samples were filtered after 1, 5, and 10 min. The high cell density and a high concentration of [14C]maltose were necessary due to the lack of thelamB gene, encoding maltoporin, in strain ED169. Maltoporin is required for the specific diffusion of maltose and maltodextrins through the outer membrane, especially at low substrate concentrations (6.Boos W. Shuman H. Microbiol. Mol. Biol. Rev. 1998; 62: 204-229Crossref PubMed Google Scholar). Cells of strain ED169 cotransformed with plasmids encoding mono-Cys variants of MalK (derivatives of pSU19) and mono-Cys variants of MalF and MalG, respectively (derivatives of pTZ18R), were grown in LB medium supplemented with ampicillin (100 μg/ml) and chloramphenicol (20 μg/ml) at 30 °C to an OD650 = 0.3. Then, 0.5 mmisopropyl-β-thiogalactoside was added and growth was continued for 2 h. Subsequently, cells were harvested, resuspended in buffer 1 (0.1 m KPi, 10 mm EDTA, pH 6.6) (8 ml/500 ml of culture), containing 0.05 mg/ml DNase I and disrupted by one passage through a French press at 27 megapascals at 4 °C. Membrane vesicles were collected by ultracentrifugation (200,000 × g for 1 h at 4 °C), resuspended in 0.5 ml of buffer 2 (0.1 m Tris-HCl, 150 mm NaCl, 10% glycerol, pH 7.0), shock frozen in liquid nitrogen, and stored at −80 °C until use. If not stated otherwise, membrane vesicles at a final concentration of 1 mg/ml in 20 mm Tris-HCl, pH 7.0, containing 5 mm MgCl2 (assay buffer) were routinely treated with CuPhe (3 mm CuSO4/9 mm1,10-phenanthroline) for 30 min on ice. The reaction was terminated by the addition of 5× SDS sample buffer, containing 5 mm N-ethylmaleimide. For studying the effect of ATP on the cross-linking reaction, membrane vesicles were first incubated in buffer 2, containing 10 mm EDTA for 30 min on ice, collected by ultracentrifugation, washed once in assay buffer containing 5 mm DTT, and finally resuspended in 20 mm Tris-HCl, pH 7.0. Cross-linking reactions were then performed as above but in the presence of 5 mm ATP. Reactions were performed under the same conditions as above but in the presence of 0.25 mm BMH, o-PDM, or p-PDM (10 mm stock solutions in dimethyl sulfoxide). After incubation for 30 min on ice, the reactions were terminated by adding 5× SDS sample buffer containing 0.5 mDTT. Standard DNA techniques were performed as in (20.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Site-directed mutagenesis was performed using Stratagene's QuikChange mutagenesis kit. The Pierce BCA microassay was used for assaying protein concentrations. SDS-PAGE and immunoblotting were performed as described in Ref. 19.Schneider E. Walter C. Mol. Microbiol. 1991; 5: 1375-1383Crossref PubMed Scopus (29) Google Scholar. Immunoblots were developed using the Western Blot Chemiluminescence Reagent Plus system (NEN Life Science Products). Antiserum against MalF was kindly provided by B. Traxler. 1,6-Bismaleimidohexane was bought from Pierce.N,N′-o-Phenylenedimaleimide andN,N′-p-phenylenedimaleimide were purchased from Sigma. As a prerequisite for the intended cross-linking experiments, we first constructed a maltose transporter devoid of the naturally occurring cysteine residues by site-directed mutagenesis. It had already been demonstrated that a Cys-less variant of MalK of S. typhimurium fully retained its function in maltose transport (16.Hunke S. Schneider E. FEBS Lett. 1999; 448: 131-134Crossref PubMed Scopus (11) Google Scholar). By using the same protocol, serine residues substituting for cysteines were introduced at positions 85, 304 and 388 in MalF, and at position 106 in MalG of E. coli, respectively. Subsequently, E. coli strain ED169, carrying a deletion of the malBregion that encodes the transport proteins, was cotransformed with plasmids pSH46 (malK798; encoding Cys-less MalK of S. typhimurium), pTAZFGQ* (encoding Cys-less MalF and Cys-less MalG of E. coli), and pTLCME (malE +; encoding wild type maltose-binding protein of E. coli). The resulting triple transformants are capable of utilizing maltose as sole source of carbon and energy, as indicated by their dark red appearance on McConkey/maltose agar (Fig. 2), thus clearly suggesting that none of the native cysteine residues of the MalFGK2 complex is essential for function. This conclusion was confirmed by transport assays, which demonstrated that the cells displayed [14C]maltose uptake at a rate corresponding to 61% of that measured with cells harboring the plasmid-borne wild type alleles (Table II).Table IIMaltose transport activities of Cys-substituted mutantsMalF/MalG variantsMalK variantsWild typeCys-lessA85CK106CV114CV117CWild type100NDNDNDNDNDCys-lessND6119555852S3C/−NDND52489361−/A3CNDND521385265−/G7CNDND71903281Cells of strain ED169 (ΔmalB107) transformed with combinations of plasmids that carry the described alleles ofmalF, malG, and malK each together with the wild type allele of malE were grown and assayed for [14C]maltose uptake as described under “Experimental Procedures.” Transport rates represent the average of at least two experiments and are given in percentage relative to that measured with control cells harboring the respective plasmid-borne wild type alleles. The 100% value corresponds to 0.04 nmol/min/109 cells. Note that the general low transport activity observed under the experimental conditions used is due to the absence of maltoporin. ND, not determined. Open table in a new tab Cells of strain ED169 (ΔmalB107) transformed with combinations of plasmids that carry the described alleles ofmalF, malG, and malK each together with the wild type allele of malE were grown and assayed for [14C]maltose uptake as described under “Experimental Procedures.” Transport rates represent the average of at least two experiments and are given in percentage relative to that measured with control cells harboring the respective plasmid-borne wild type alleles. The 100% value corresponds to 0.04 nmol/min/109 cells. Note that the general low transport activity observed under the experimental conditions used is due to the absence of maltoporin. ND, not determined. The data also demonstrate that a functional transport complex can be assembled from mutant subunits of different organisms. This is consistent with the observation that wild type MalK of S. typhimurium can fully substitute for E. coli MalK in maltose transport (10.Wilken S. Schmees G. Schneider E. Mol. Microbiol. 1996; 22: 555-666Crossref PubMed Scopus (112) Google Scholar, 21.Dahl M.K. Francoz E. Saurin W. Boos W. Manson M.D. Hofnung M. Mol. Gen. Genet. 1989; 218: 199-207Crossref PubMed Scopus (51) Google Scholar). Thus, the hybrid transport complex represents a well suited model system for the intended study. Starting from the Cys-less variants, we next constructed mono-Cys derivatives of each subunit by site-directed mutagenesis, using plasmids pSH30 and pTAZFGQ* as templates. In MalK, cysteines were introduced in place of Ala-85, Lys-106, Val-114, and Val-117, respectively, which by genetic and biochemical approaches had been identified as putative candidates for interaction with MalF and/or MalG (10.Wilken S. Schmees G. Schneider E. Mol. Microbiol. 1996; 22: 555-666Crossref PubMed Scopus (112) Google Scholar, 12.Mourez M. Jéhano M. Schneider E. Dassa E. Mol. Microbiol. 1998; 30: 353-363Crossref PubMed Scopus (33) Google Scholar, 13.Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar). In MalF and MalG, we introduced cysteine residues in the respective EAA loops at those positions that, when mutated, gave rise to the described suppressor mutations in malK (13.Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar). Accordingly, Glu-401 (E1) in MalF, and Glu-190 (E1), Ala-192 (A3), and Gly-196 (G7) in MalG, respectively, were individually substituted for by cysteines. In addition, Ser-403 (S3) in MalF, which was shown previously to be less sensitive to replacements (13.Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar), was also modified. From here on, the mono-Cys variants are denoted by a set of two symbols separated by a slash (13.Mourez M. Hofnung M. Dassa E. EMBO J. 1997; 16: 3066-3077Crossref PubMed Scopus (165) Google Scholar). The first symbol describes cysteine replacements in MalF and the second those in MalG. A non-mutated (Cys-less) variant is represented by a dash. Numbers between letters point to the relative position of the residue in the consensus sequence (see Fig.1 B). Plasmids carrying Cys substitutions in MalK (all derivatives of pSU19, see Table I) and plasmids carrying Cys substitutions in either MalF or MalG, were cotransformed together with pTLCME (malE +) into strain ED169. Additionally, combinations of plasmids carrying Cys substitutions of MalK and Cys-less MalF/MalG or vice versa were also introduced into ED169. Growth of the transformants were again tested on McConkey/maltose agar. As shown in Fig.2, all mono-Cys variants of MalK when combined with MalF(−)/MalG(−), MalF(S3C)/MalG(−) or MalF(−)/MalG(G7C) supported growth on maltose comparable to the Cys-less complex. Some reduction in growth, as indicated by a less intense red color of the wells, was observed in combination with MalF(−)/MalG(A3C). These data were largely confirmed by measuring initial rates of [14C]maltose uptake. As summarized in Table II, the majority of Cys-substituted mutant strains exhibited transport rates of at least 48% of those measured with wild type cells. Only two combinations (MalKA85C with MalF(−)/MalG(−) and MalKV114C with MalF(−)/MalG(G7C)) gave lower values. Transformants carrying MalF(E1C)/MalG(−) and MalF(−)/MalG(E1C) behaved differently. As indicated by the almost colorless wells in Fig.2, substitution of cysteine for glutamate at position 1 in MalF strongly affected growth in a complex with Cys-less MalK or any of the mono-Cys MalK variants. However, in some experiments, newly transformed cells also appeared as red colonies on MacConkey/maltose plates. The reasons for these inconsistent results are not known. In contrast, cysteine replacing glutamate at position 1 in MalG consistently allowed growth when combined with either Cys-less MalK or MalKV114C, while other complexes failed to support growth on maltose. For cells containing the MalKV117C variant, this result was confirmed by the absence of any measurable maltose transport activity (data not shown). Thus, in order to exclude potential artifacts created by nonfunctional transport complexes, combinations with Cys substitutions at position 1 in both MalF and MalG were eliminated from subsequent cross-linking experiments. The initial screening experiments for MalK-MalF and MalK-MalG cross-link formation were performed with inside-out membrane vesicles containing combinations of MalKA85C with MalF(S3C)/MalG(−) and MalF(−)/MalG(A3C), respectively. Strong cross-links were observed in both cases on immunoblots probed with antiserum to MalK after CuPhe treatment for 30 min on ice (Fig. 3,A and B, lanes 2). In contrast, when CuPhe-treated control samples were electrophoresed in the presence of DTT, no cross-links were detected (data not shown). The identity of the putative FK and GK products was verified by re-probing the blots with antisera raised against MalF and MalG, respectively. As demonstrated in Fig. 3 (panels A andB, lanes 4), cross-links with molecular masses similar to those detected with anti-MalK antiserum were observed in the presence of CuPhe only. Since both antisera cross-reacted with other proteins, in subsequent experiments, immunoblots were probed with the anti-MalK antiserum only. An additional, slower migrating band (∼97 kDa) was picked up by the MalK antiserum in the combination with MalF(−)/MalG(A3C) (Fig.3 B, lane 2). A weak reaction at about the same position was also observed with the anti-MalG antiserum (Fig.3 B, lanes 3 and 4); however, independent of the presence of CuPhe, which is in contrast to the result obtained with the antiserum to MalK. Thus, it is concluded that the bands seen in Fig. 3 B (lanes 3 and 4) are due to an unspecific cross-reaction and that the cross-link detected with anti-MalK antiserum" @default.
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