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.

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Figures

FIG. 1.

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.

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.

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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References

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