Direct demonstration of ATP-dependent release of SecA from a translocating preprotein by surface plasmon resonance.
Translocase mediates the transport of preproteins across the inner membrane of Escherichia coli. SecA binds with high affinity to the membrane-embedded protein-conducting SecYEG complex and serves as both a receptor for secretory proteins and as an ATP-driven molecular motor. Cycles of ATP binding and hydrolysis by SecA drive the progressive movement of the preprotein across the membrane. Surface plasmon resonance allows an online monitoring of protein interactions. Here we report on the kinetic analysis of the interaction between SecA and the membrane-embedded SecYEG complex. Immobilization of membrane vesicles containing overproduced SecYEG on the Biacore Pioneer L1 chip allows the detection of high affinity SecA binding to the SecYEG complex and online monitoring of the translocation of the secretory protein proOmpA. SecA binds tightly to the SecYEG.proOmpA complex and is released only upon ATP hydrolysis. The results provide direct evidence for a model in which SecA cycles at the SecYEG complex during translocation.
de Keyzer
Jeanine
J
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
van der Does
Chris
C
Kloosterman
Tomas G
TG
Driessen
Arnold J M
AJ
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D020349:Surface Plasmon Resonance; D013997:Time Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C088616:SecG protein, E coli; C057841:SecY protein, E coli; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins