SECY AND SECA INTERACT TO ALLOW SECA INSERTION AND PROTEIN TRANSLOCATION ACROSS THE ESCHERICHIA-COLI PLASMA-MEMBRANE

SecA, the preprotein-driving ATPase in Escherichia coli, was shown pre viously to insert deeply into the plasma membrane in the presence of A TP and a preprotein; this movement of SecA was proposed to be mechanis tically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane-embedded heterotr imeric complex, in the SecA insertion reaction. We identified a secY m utation (secY205), affecting the most carboxy-terminal cytoplasmic dom ain, that did not allow ATP and preprotein-dependent productive SecA i nsertion. while allowing idling insertion without the preprotein. Thus , the secY205 mutation might affect the SecYEG 'channel' structure in accepting the preprotein-SecA complex or its opening by the complex. W e isolated secA mutations that allele-specifically suppressed the secY 205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was pur ified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein co uld also insert into the mutant membrane vesicles in vitro. These resu lts provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in re sponse to a preprotein and ATP and suggest that SecA drives protein tr anslocation by inserting into the membrane in vivo.