Covalently dimerized SecA is functional in protein translocation.
The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction.
de Keyzer
Jeanine
J
Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute and Materials and Science Centre Plus, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
van der Sluis
Eli O
EO
Spelbrink
Robin E J
RE
Nijstad
Niels
N
de Kruijff
Ben
B
Nouwen
Nico
N
van der Does
Chris
C
Driessen
Arnold J M
AJ
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry*; D009154:Mutation; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014508:Urea / Q000494:pharmacology
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; D003545:Cysteine; D010100:Oxygen