Tyr‐326 plays a critical role in controlling SecA–preprotein interaction

Abstract

SecA is an essential ATP‐dependent motor protein that interacts with the preprotein and translocon to drive protein translocation across the eubacterial plasma membrane. A region containing residues 267–340 has been proposed to comprise the preprotein binding site of Escherichia coli SecA. To elucidate the function of this region further, we isolated mutants using a combination of region‐specific polymerase chain reaction (PCR) mutagenesis and a genetic and biochemical screening procedure. Although this region displayed considerable plasticity based on phylogenetic and genetic analysis, Tyr‐326 was found to be critical for SecA function. secA mutants with non‐conservative substitutions at Tyr‐326 showed strong protein secretion defects in vivo and were completely defective for SecA‐dependent translocation ATPase activity in vitro . The SecA‐Y326 mutant proteins were normal in their membrane, SecYE and nucleotide‐binding properties. However, they exhibited a reduced affinity for preprotein and were defective in preprotein release, as assessed by several biochemical assays. Our results indicate that the region containing Tyr‐326 functions as a conformational response element to regulate the preprotein binding and release cycle of SecA.