TOPOLOGY OF THE INTEGRAL MEMBRANE FORM OF ESCHERICHIA-COLI SECA PROTEIN REVEALS MULTIPLE PERIPLASMICALLY EXPOSED REGIONS AND MODULATION BY ATP BINDING

SecA insertion and integration into the Escherichia coli inner membran e is a critical step for the catalysis of protein translocation across this layer, To understand this step further, SecA topology was invest igated, To determine which regions of SecA are periplasmically exposed , right-side out membrane vesicles were prepared from strains synthesi zing monocysteine SecA variants produced by mutagenesis and probed wit h a membrane-impermeant sulfhydryl-labeling reagent, To determine whic h regions of SecA contain membrane-integration determinants, inverted inner membrane vesicles were subjected to proteolysis, and integral-me mbrane fragments of SecA were identified with region-specific antibodi es, The membrane association properties of various truncated SecA spec ies 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 w here portions are periplasmically accessible, Furthermore, the inserti on and penetration of the amino-terminal third of SecA, which includes the proposed preprotein binding domain, is subject to modulation by A TP binding, The importance of these studies to the cycle of membrane i nsertion and de-insertion of SecA that promotes protein translocation and SecA's proximity to the preprotein channel are discussed.