Topologically fixed SecG is fully functional - PubMed (original) (raw)
Topologically fixed SecG is fully functional
Eli O van der Sluis et al. J Bacteriol. 2006 Feb.
Abstract
It has been proposed that the bitopic membrane protein SecG undergoes topology inversion during translocation of (pre)proteins via SecYEG. Here we show that SecG covalently cross-linked to SecY cannot invert its topology while remaining fully functional in protein translocation. Our results strongly disfavor topology inversion of SecG during protein translocation.
Figures
FIG. 1.
Highly efficient disulfide cross-linking of SecY(T179C) to SecG(K26C). NN104-derived IMVs overexpressing different combinations of SecY (SecE) and SecG or the empty expression vector (lanes 1) were oxidized with Na2S4O6 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie brilliant blue staining (A), anti-SecY (B), or anti-SecG immunodetection (17) (C) according to standard procedures. Bands corresponding to SecY, SecG, dimeric SecG (SecG2), and the SecY-SecG cross-link product are labeled correspondingly, and the weak band indicated with an asterisk represents SecG cross-linked to the N-terminal proteolytic fragment of SecY (23). For quantitation of the SecY-SecG cross-linking efficiency, the Coomassie brilliant blue-stained gel was imaged and analyzed. Note that no quantitative information can be gained from the Western blots due to the reduced blotting efficiency of the cross-linked adducts.
FIG. 2.
SecG cross-linked to SecY does not invert its membrane topology. Oxidized IMVs overexpressing SecY(T179C)EG(K26C) (panels 1 and 2) or wild-type SF100 IMVs (panels 3) were subjected to the SecG topology inversion assay (A: −ATP plus AMP-PNP; B: complete plus AMP-PNP; see text for details) as described before (18) under nonreducing conditions. Samples were analyzed by nonreducing (panels 1) or reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (panels 2 and 3) followed by immunodetection with antibodies raised against the extreme C terminus of SecG (17). Bands corresponding to the SecY-SecG cross-link product, SecG, dimeric SecG (SecG2), and the 9-kDa C-terminal fragment of SecG, are indicated. The applied concentrations of proteinase K (PK) are indicated at the bottom of each panel. Note that a small amount of the 9-kDa fragment is always observed upon overexpression of SecG (18).
FIG. 3.
Cross-linked SecY(T179C)EG(K26C) is as active as wild-type SecYEG. Oxidized IMVs overexpressing the indicated SecYEG complexes (see legend to Fig. 1) were analyzed for in vitro translocation of fluorescein maleimide-labeled pro-OmpA(C302S) under nonreducing conditions (A) or in the presence of 5 mM dithiothreitol (DTT) (B) as described (23). Cysteineless SecYEG has previously been shown to be as active as wild-type SecYEG (10). (C) Oxidized IMVs overexpressing SecYEG (open symbols) or SecY(T179C)EG(K26C) (solid symbols) were analyzed for pro-OmpA-stimulated SecA ATPase activity under nonreducing conditions as described (4) with the indicated amounts of SecA.
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