Topology of the integral membrane form of Escherichia coli SecA protein reveals multiple periplasmically exposed regions and modulation by ATP binding.
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.
Ramamurthy
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Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459, USA.
Oliver
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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
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