Abstract
SecA insertion and integration into the Escherichia coli inner membrane is a critical step for the catalysis of protein translocation across this layer. To understand this step further, SecA topology was investigated. To determine which regions of SecA are periplasmically exposed, right-side out membrane vesicles were prepared from strains synthesizing monocysteine SecA variants produced by mutagenesis and probed with a membrane-impermeant sulfhydryl-labeling reagent. To determine which regions of SecA contain membrane-integration determinants, inverted inner membrane vesicles were subjected to proteolysis, and integral-membrane fragments of SecA were identified with region-specific antibodies. The membrane association properties of various truncated SecA species produced in vivo were also determined. Our analysis indicates that the membrane topology of SecA is complex with amino-terminal, central, and carboxyl-terminal regions of SecA integrated into the membrane where portions are periplasmically accessible. Furthermore, the insertion and penetration of the amino-terminal third of SecA, which includes the proposed preprotein-binding domain, is subject to modulation by ATP binding. The importance of these studies to the cycle of membrane insertion and de-insertion of SecA that promotes protein translocation and SecA's proximity to the preprotein channel are discussed.
Highlights
The translocation ATPase, and the heterotrimeric SecYEG protein, which appears to constitute the preprotein channel based on recent phylogenetic evidence (6 –10)
It has been proposed that the topology inversion of SecG protein that was observed during protein translocation may enhance the membrane cycling behavior of SecA protein (25)
In this study we have utilized a combination of site-directed sulfhydryl labeling, proteolysis, or truncation of SecA to develop a map of the membrane topology of this protein
Summary
Plasmids, Media, and Chemicals—BL21.19 (secA13 (Am) supF (Ts) trp (Am) zch::Tn10 recA::CAT clpA::KAN) (27) is a derivative of BL21 (DE3) (28) that was used as the host strain for plasmids. Biotinylation of RSO and Western Blotting—RSO (200 g of membrane protein per sample) were sedimented at 12,000 ϫ g for 10 min, and Cys residues were reduced by resuspension of RSO in 200 l of 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM DTT followed by incubation for. MPB dissolved in dimethyl sulfoxide (50 mg mlϪ1) was added to RSO to a final concentration of 0.05 mM, followed by incubation at 0 °C for 3 min. RSO were collected by sedimentation and resuspended in 200 l of lysis buffer (10 mM Tris-HCl, pH 8.0, 1% SDS, 1 mM EDTA), followed by immunoprecipitation with either 2 l of anti-SecA antisera or 2 l of anti-FrD-A antisera as described previously (36). Assays for SecA-dependent endogenous, membrane, and translocation ATPase activities utilizing the colorimetric reagent malachite green were performed as described previously (27)
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