Increases in acidic phospholipid contents specifically restore protein translocation in a cold-sensitive secA or secG null mutant.
Both the secAcsR11 and DeltasecG::kan mutations cause cold-sensitive growth, although the growth defect due to the latter mutation occurs in a strain-specific manner. Overexpression of pgsA encoding phosphatidylglycerophosphate synthase suppresses the growth defects of the two mutants. We investigated the mechanism underlying the pgsA-dependent suppression of the two mutations using purified mutant SecA and inverted membrane vesicles (IMVs) prepared from pgsA-overexpressing cells. The acidic phospholipid content increased by about 10% upon pgsA overexpression. This increase resulted in the stimulation of proOmpA translocation only when mutant SecA or SecG-depleted IMVs were used. The translocation-coupled ATPase activity of SecA was significantly defective with the mutant SecA or SecG-depleted IMVs, but it recovered to a near normal level when the acidic phospholipid level was increased. The stimulation of ATPase activity was observed only at low temperature. The steady-state level of membrane-inserted SecA was low with the mutant SecA or SecG-depleted IMVs, and it decreased further upon the increase in the acidic phospholipid content. However, the level of SecA insertion markedly increased upon the inhibition of SecA deinsertion by the addition of beta,gamma-imido adenosine 5'-triphosphate (AMP-PNP), especially with IMVs containing increased levels of acidic phospholipids. These results indicate that the increase in the level of acidic phospholipids stimulates the SecA cycle in the two mutants by facilitating both the insertion and deinsertion of SecA.
Suzuki
H
H
Institute of Molecular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Nishiyama
K
K
Tokuda
H
H
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D010743:Phospholipids / Q000378:metabolism*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010743:Phospholipids; D000069816:SEC Translocation Channels; C499394:SecD protein, E coli; C065493:SecE protein, E coli; C489583:SecF protein, E coli; C088616:SecG protein, E coli; C057841:SecY protein, E coli; C075767:secD protein, Bacteria; C075768:secF protein, Bacteria; D017855:Transferases (Other Substituted Phosphate Groups); C020676:CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins