Novel translocation intermediate allows re-evaluation of roles of ATP, proton motive force and SecG at the late stage of preprotein translocation.
Presecretory proteins such as pOmpA are translocated across the inner membrane of Escherichia coli by Sec translocase powered by ATP and proton motive force (PMF). Translocation activity has been determined by protease protection assaying in vitro. We identified a new translocation intermediate at a late stage, which was protected by proteinase K (PK), but became PK sensitive upon urea extraction. At a late stage of pOmpA translocation driven by PMF in the presence of a nonhydrolyzable ATP analogue, the PK-protected materials arose, but were pulled back upon urea extraction, indicating that completion of translocation requires ATP hydrolysis. When inverted membrane vesicles prepared from secG-null strain (ΔSecG IMV) were used in the absence of PMF, the translocation intermediate was accumulated. When the ATP concentration was low in the absence of PMF, the translocation intermediate was also accumulated. Imposition of PMF in the presence of a low ATP concentration caused recovery of pOmpA translocation and resistance to urea extraction for SecG+IMV, but not for ΔSecG IMV. Thus, analysis of the late translocation intermediate showed that two of three constituents, physiological concentration of ATP, PMF and SecG, are required for the catalytic cycle of preprotein translocation, that is, completion and subsequent initiation of translocation.
Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Morioka, 020-8550, Japan.|Department of Biological Chemistry and Food Science, Faculty of Agriculture, Iwate University, Morioka, 020-8550, Japan.
Ken-Ichi
KI
Nishiyama
Faculty of Nutritional Sciences, The University of Morioka, Takizawa, 020-0694, Japan.
Hajime
H
Tokuda
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D021381:Protein Transport; D018892:Proton-Motive Force; D000069816:SEC Translocation Channels / Q000378:metabolism*; D014508:Urea / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C088616:SecG protein, E coli; C078167:OMPA outer membrane proteins; D000255:Adenosine Triphosphate; D014508:Urea