title: Mapping polypeptide interactions of the SecA ATPase during translocation.
abstract: Many bacterial proteins, including most secretory proteins, are translocated across the plasma membrane by the interplay of the cytoplasmic SecA ATPase and a protein-conducting channel formed by the SecY complex. SecA catalyzes the sequential movement of polypeptide segments through the SecY channel. How SecA interacts with a broad range of polypeptide segments is unclear, but structural data raise the possibility that translocation substrates bind into a "clamp" of SecA. Here, we have used disulfide bridge cross-linking to test this hypothesis. To analyze polypeptide interactions of SecA during translocation, two cysteines were introduced into a translocation intermediate: one that cross-links to the SecY channel and the other one for cross-linking to a cysteine placed at various positions in SecA. Our results show that a translocating polypeptide is indeed captured inside SecA's clamp and moves in an extended conformation through the clamp into the SecY channel. These results define the polypeptide path during SecA-mediated protein translocation and suggest a mechanism by which ATP hydrolysis by SecA is used to move a polypeptide chain through the SecY channel.
authors.affiliation: Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Benedikt W
authors.initials: BW
authors.lastname: Bauer
authors.forename: Tom A
authors.initials: TA
authors.lastname: Rapoport
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping; D011498:Protein Precursors / Q000378:metabolism; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000187:drug effects; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D010455:Peptides; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; C057184:outer membrane protein A precursor (E coli); D013762:Tetrahydrofolate Dehydrogenase; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins