Dimeric SecA is essential for protein translocation.
SecA facilitates bacterial protein translocation by its association with presecretory or membrane proteins and the SecYEG translocon channel. Once assembled, SecA ATPase undergoes cycles of membrane insertion and retraction at SecYEG that drive protein translocation in a stepwise fashion. SecA exists in equilibrium between a monomer and dimer, and association with its translocation ligands shifts this equilibrium dramatically. Here, we examined the proposal that protein translocation can occur by means of a SecA monomer. We produced a mutant SecA protein lacking residues 2-11, which was found to exist mostly as a monomer, and it was unable to complement a conditional-lethal secA mutant, was inactive for in vitro protein translocation, and was poorly active for translocation ATPase activity. Furthermore, we developed a technique termed membrane trapping, where wild-type SecA subunits became trapped within the membrane by overproduction of membrane-stuck mutant SecA proteins, and, in one case, a membrane-associated SecA heterodimer was demonstrated. Finally, we examined both endogenous and reconstituted membrane-bound SecA and found a significant level of SecA dimer in both cases, as assessed by chemical crosslinking. Collectively, our results strongly suggest that membrane-bound SecA dimer is critical for the protein translocation cycle, although these results cannot exclude participation of SecA monomer at some stage in the translocation process. Our findings have important implications regarding SecA motor function and translocon assembly and activation.
Jilaveanu
Lucia B
LB
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459, USA.
Zito
Christopher R
CR
Oliver
Donald
D
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D015153:Blotting, Western; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D008024:Ligands; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins