title: Topology of the integral membrane form of Escherichia coli SecA protein reveals multiple periplasmically exposed regions and modulation by ATP binding.
abstract: SecA insertion and integration into the Escherichia coli inner membrane is a critical step for the catalysis of protein translocation across this layer. To understand this step further, SecA topology was investigated. To determine which regions of SecA are periplasmically exposed, right-side out membrane vesicles were prepared from strains synthesizing monocysteine SecA variants produced by mutagenesis and probed with a membrane-impermeant sulfhydryl-labeling reagent. To determine which regions of SecA contain membrane-integration determinants, inverted inner membrane vesicles were subjected to proteolysis, and integral-membrane fragments of SecA were identified with region-specific antibodies. The membrane association properties of various truncated SecA species produced in vivo were also determined. Our analysis indicates that the membrane topology of SecA is complex with amino-terminal, central, and carboxyl-terminal regions of SecA integrated into the membrane where portions are periplasmically accessible. Furthermore, the insertion and penetration of the amino-terminal third of SecA, which includes the proposed preprotein-binding domain, is subject to modulation by ATP binding. The importance of these studies to the cycle of membrane insertion and de-insertion of SecA that promotes protein translocation and SecA's proximity to the preprotein channel are discussed.
authors.affiliation: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
authors.forename: V
authors.initials: V
authors.lastname: Ramamurthy
authors.forename: D
authors.initials: D
authors.lastname: Oliver
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C461599:maltose transport system, E coli; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; D003545:Cysteine