Membrane topology inversion of SecG detected by labeling with a membrane-impermeable sulfhydryl reagent that causes a close association of SecG with SecA.
SecG stimulates protein translocation in Escherichia coli by facilitating the membrane insertion-deinsertion cycle of SecA. SecG was previously shown to undergo membrane topology inversion, since SecA-dependent protein translocation renders the membrane-protected region of SecG sensitive to external proteases. To examine this topology inversion in more detail without protease-treatment, SecG derivatives with a single cysteine residue at various positions were labeled in the presence and absence of protein translocation with a membrane impermeable SH reagent, 4-acetamido-4'-maleimidylstilbene-2-2'-disulfonic acid (AMS). Treatment of spheroplasts with AMS revealed that a cysteine residue in the cytoplasmic region of SecG could be labeled from the periplasm side only in the presence of protein translocation, whereas a cytoplasmic protein, elongation factor, Tu, remained unlabeled. Treatment of inverted membrane vesicles with AMS also revealed that cysteine residues in the periplasmic region were labeled from the cytoplasmic side of membranes only when protein translocation was in progress. This labeling required ATP, SecA and a precursor protein, and became more efficient as the position of the cysteine residue became closer to the C-terminus. Crosslinking analyses revealed that the interaction between SecG and SecA in membranes markedly increases when SecA and SecG undergo membrane-insertion and topology inversion, respectively. Thus, the two most dynamic components of the translocation machinery were found for the first time to interact with each other when both undergo conformational changes.
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002463:Cell Membrane Permeability; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013104:Spheroplasts / Q000737:chemistry / Q000378:metabolism; D013267:Stilbenes / Q000737:chemistry* / Q000494:pharmacology*; D013451:Sulfonic Acids / Q000737:chemistry* / Q000494:pharmacology*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C088616:SecG protein, E coli; D013267:Stilbenes; D013451:Sulfonic Acids; C057184:outer membrane protein A precursor (E coli); C079532:4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D010444:Peptide Elongation Factor Tu; D000081416:SecA Proteins; D003545:Cysteine