INTEGRATION OF SECA PROTEIN INTO THE ESCHERICHIA-COLI INNER MEMBRANE IS REGULATED BY ITS AMINO-TERMINAL ATP-BINDING DOMAIN

SecA protein, the ATPase promoting translocation of proteins across th e Escherichia coli inner membrane, contains two ATP-binding domains th at differ greatly in their affinity for bound nucleotide. In order to define more precisely the location of the high-affinity nucleotide-bin ding site, oligonucleotide-directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine-specific clea vage reagent was employed to generate defined peptides of SecA protein after photocross-linking with [alpha-P-32]-ATP, This analysis reveale d that the nucleotide was crosslinked between amino acid residues 75 a nd 97 of SecA protein. The biochemical function of the high affinity A TP-binding domain was explored by subcellular fractionation studies wh ich demonstrated that SecA proteins defective in this region were foun d almost exclusively in their integral membrane form, while SecA prote ins with defects in the low-affinity ATP-domain showed a normal distri bution of cytosolic, peripheral and integral membrane forms. Interesti ngly, the SecA51(Ts) protein that has a Leu to Pro substitution at ami no acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of pr oteins with defects in the high-affinity ATP-binding site. These resul ts delimit more precisely the high-affinity ATP-binding domain of SecA , indicate the importance of the early amino-terminal region of SecA p rotein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the inner m embrane. Our results lead to a simple model for the regulation of a cy cle of SecA insertion into, and de-insertion from, the inner membrane by the activity of the high-affinity ATP-binding domain.