Topological and mutational analysis of KpsM, the hydrophobic component of the ABC-transporter involved in the export of polysialic acid in Escherichia coli K1.
The 17 kb kps gene cluster of Escherichia coli K1, which encodes the information required for synthesis, assembly and translocation of the polysialic acid capsule of E. coli K1, is divided into three functional regions. Region 3 contains two genes, kpsM and kpsT, essential for the transport of capsule polymer across the cytoplasmic membrane. The hydrophobicity profile of KpsM suggests that it is an integral membrane protein while KpsT contains a consensus ATP-binding site. KpsM and KpsT belong to the ATP-binding cassette (ABC) superfamily of membrane transporters. In this study, we investigate the topology of KpsM within the cytoplasmic membrane using beta-lactamase fusions and alkaline phosphatase sandwich fusions. Our analysis provides evidence for a model of KpsM having six membrane-spanning regions, with the N- and C-terminal domains facing the cytoplasm, and a short domain within the third periplasmic loop, which we refer to as the SV-SVI linker localizing in the membrane. Protease digestion studies are consistent with regions of KpsM exposed to the periplasmic space. In vivo cross-linking studies provide support for dimerization of KpsM within the cytoplasmic membrane. Linker-insertion and site-directed mutagenesis define the N-terminus, the first cytoplasmic loop, and the SV-SVI linker as regions that are important for the function of KpsM in K1 polymer transport.
Pigeon
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RP
Department of Microbiology and Immunology, University of Rochester Medical Center, New York 14642.
Silver
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RP
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001693:Biological Transport, Active; D003432:Cross-Linking Reagents; D018076:DNA, Complementary / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012794:Sialic Acids / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D012794:Sialic Acids; C021319:polysialic acid; C068104:kpsM protein, E coli; D010450:Endopeptidases