2 DISTINCT ATP-BINDING DOMAINS ARE NEEDED TO PROMOTE PROTEIN EXPORT BY ESCHERICHIA-COLI SECA ATPASE

Six putative ATP-binding motifs of SecA protein were altered by oligon ucleotide-directed mutagenesis to try to define the ATP-binding region s of this multifunctional protein. The effects of the mutations were a nalysed by genetic and biochemical assays. The results show that SecA contains two essential ATP-binding domains. One domain is responsible for high-affinity ATP binding and contains motifs A0 and BO, located a t amino acid residues 102-109 and 198-210, respectively. A second doma in is responsible for low-affinity ATP binding and contains motifs A3 and a predicted B motif located at amino acid residues 503-511 and 631 -653, respectively. The ATP-binding properties of both domains were es sential for SecA-dependent translocation ATPase and in vitro protein t ranslocation activities. The significance of these findings for the me chanism of SecA-dependent protein translocation is discussed.