PROTEIN TRANSLOCATION FUNCTIONS OF ESCHERICHIA-COLI SECY - IN-VITRO CHARACTERIZATION OF COLD-SENSITIVE SECY MUTANTS

Protein translocation across the plasma membrane of E coli is facilita ted by Sec factors, including the membrane-embedded SecYEG subunit and the SecA ATPase. Although there is complete agreement that SecA is es sential for protein translocation, some publications question the esse ntialness of SecY. We previously isolated a number of cold-sensitive m utants of secY and characterized their in vivo phenotypes. In this stu dy, we characterized membrane vesicles prepared from these mutants wit h respect to their in vitro activities to support protein translocatio n and to activate the SecA ATPase. These studies revealed several sing le amino acid alterations that abolish these in vitro activities of me mbrane vesicles. In particular, several mutations in the two most carb oxy-terminal cytoplasmic domains of SecY prevented SecA from functioni ng as the translocation ATPase. A number of mutants showed strong corr elations between in vivo protein export ability, in vitro translocatio n activity and in vitro translocation ATPase activity, substantiating the importance of SecY irt vivo and in vitro. A few other mutants were affected in only one or two aspects of these properties, suggesting t hat they were differentially affected in some substeps of translocatio n. These results provide further evidence that SecY has vital roles in protein translocation, in which the 'motor' function of SecA and the 'channel' function of SecYEG should be coordinated.