Syd, a SecY-interacting protein, excludes SecA from the SecYE complex with an altered SecY24 subunit.
Syd is an Escherichia coli cytosolic protein that interacts with SecY. Overproduction of this protein causes a number of protein translocation-related phenotypes, including the strong toxicity against the secY24 mutant cells. Previously, this mutation was shown to impair the interaction between SecY and SecE, the two fundamental subunits of the membrane-embedded part of protein translocase. We have now studied in vitro the mechanisms of the Syd-directed inhibition of protein translocation. Pro-OmpA translocation into inverted membrane vesicles (IMVs) prepared from the secY24 mutant cells as well as the accompanied translocation ATPase activity of SecA were rapidly inhibited by purified Syd protein. In the course of protein translocation, high affinity binding of preprotein-bearing SecA to the translocase on the IMV is followed by ATP-driven insertion of the 30-kDa SecA segment into the membrane. Our experiments using 125I-labeled SecA and the secY24 mutant IMV showed that Syd abolished both the high affinity SecA binding and the SecA insertion. Syd was even able to release the inserted form of SecA that had been stabilized by a nonhydrolyzable ATP analog. Syd affected markedly the proteolytic digestion pattern of the IMV-integrated SecY24 protein, suggesting that Syd exerts its inhibitory effect by interacting directly with the SecY24 protein. In accordance with this notion, a SecY24 variant with a second site mutation (secY249) resisted the Syd action both in vivo and in vitro. Thus, Syd acts against the SecY24 form of translocase, in which SecY-SecE interaction has been compromised, to exclude the SecA motor protein from the SecYE channel complex.
Matsuo
E
E
Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
Mori
H
H
Shimoike
T
T
Ito
K
K
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000483:Alleles; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D010458:Peptidyl Transferases / Q000037:antagonists & inhibitors; D011498:Protein Precursors / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C057841:SecY protein, E coli; C092505:Syd protein, E coli; C057184:outer membrane protein A precursor (E coli); D010458:Peptidyl Transferases; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins