Preprotein Binding by ATP-Binding Site Mutants of the Bacillus Subtilis SecA

The preprotein translocase of Escherichia coli (Wickner et al.,1991) guards preproteins from the site of synthesis at the ribosome in the cytosol to the processed form to be released into the periplasm. SecA (Schmidt et al.,1988) is the ATP-hydrolysing, peripheral subunit of the translocase (Brundage et al.,1990; Wickner et al., 1991), and plays an essential role in preprotein translocation (Lill et al., 1989). The low endogenous ATPase activity of SecA is stimulated by interactions with acidic phospholipids, preproteins and the SecY/E protein (Lill et al.,1989, 1990; Brundage et al., 1990). This stimulated ATPase activity initiates translocation which is further driven by ATP hydrolysis and Ap (Schiebel et al., 1991; Driessen, 1992). Biochemical studies suggest that SecA possess three ATP binding sites (Lill et al., 1989; Oliver, 1993). Only one domain shows a significant level of sequence similarity to the Walker A- and B-motifs for a NTP-binding site (Walker et al., 1982) (Fig. 1). Both regions are highly conserved among different bacterial and algal SecA homologues. To analyze the function of this putative ATP binding site, we started a site-directed mutagenesis approach and changed critical residues of the A-domain (Klose et al., 1983; van der Wolk et al., 1993) of the Bacillus subtilis SecA homolog (Overhoff et al., 1991). Now we report on the localization of the B-domain and further characterized ATP- and preprotein-binding activities of the mutants.