Short hydrophobic segments in the mature domain of ProOmpA determine its stepwise movement during translocation across the cytoplasmic membrane of Escherichia coli.
Based on the finding that a series of engineered proOmpAs containing disulfide-bridged loops of different sizes at different positions exhibits a discontinuous mode of polypeptide transit across the cytoplasmic membrane of Escherichia coli, we suggested previously that the translocation of preproteins takes place at every 30 amino acid residues (Uchida, K., Mori, H., and Mizushima, S. (1995) J. Biol. Chem. 270, 30862-30868). In the present study, we investigated the molecular mechanism underlying this stepwise translocation. Deletion or relocation of hydrophobic segments of the mature domain of proOmpA (H1, residues 233-237; H2, residues 261-265) significantly altered the pattern of the stepwise translocation. The stepwise mode of polypeptide insertion was also observed with reconstituted proteoliposomes comprising purified SecA, SecY, and SecE. Cross-linking experiments involving a photoactivable cross-linker revealed that SecY and SecA are the components which interact with the hydrophobic segment of proOmpA. The present results indicate that the hydrophobic segments of the mature domains of preproteins interact with membrane embedded translocase during polypeptide transit across the membrane, which causes a discontinuous mode of polypeptide movement.
Sato
K
K
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226, Japan.
Mori
H
H
Yoshida
M
M
Tagaya
M
M
Mizushima
S
S
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry*; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C057184:outer membrane protein A precursor (E coli); D019286:Endopeptidase K; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins