Bacterial Sec protein transport is rate-limited by precursor length: a single turnover study.
An in vitro real-time single turnover assay for the Escherichia coli Sec transport system was developed based on fluorescence dequenching. This assay corrects for the fluorescence quenching that occurs when fluorescent precursor proteins are transported into the lumen of inverted membrane vesicles. We found that 1) the kinetics were well fit by a single exponential, even when the ATP concentration was rate-limiting; 2) ATP hydrolysis occurred during most of the observable reaction period; and 3) longer precursor proteins transported more slowly than shorter precursor proteins. If protein transport through the SecYEG pore is the rate-limiting step of transport, which seems likely, these conclusions argue against a model in which precursor movement through the SecYEG translocon is mechanically driven by a series of rate-limiting, discrete translocation steps that result from conformational cycling of the SecA ATPase. Instead, we propose that precursor movement results predominantly from Brownian motion and that the SecA ATPase regulates pore accessibility.
Liang
Fu-Cheng
FC
Department of Molecular and Cellular Medicine, College of Medicine, The Texas A&M Health Science Center, College Station, TX 77843, USA.
Bageshwar
Umesh K
UK
Musser
Siegfried M
SM
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009532:Nickel / Q000494:pharmacology; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013050:Spectrometry, Fluorescence; D019802:Succinic Acid / Q000494:pharmacology; D013997:Time Factors
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C057184:outer membrane protein A precursor (E coli); D000266:Adenylyl Imidodiphosphate; D009532:Nickel; D000255:Adenosine Triphosphate; D019802:Succinic Acid; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins