Cellular systems that control protein dynamism across the membrane

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Using a model organism E. coli, we have investigated the cellular systems that mediate proper biogenesis of proteins, especially those integrated into and transported across membranes. The targets of our analyses included the Sec machinery that allows protein translocation and integration, membrane-associated proteases that are important in membrane protein quality control as well as envelope stress responses, and the Dsb system that introduces disulfide bonds into proteins. We have determined crystal structures of the SecA translocation motor and the SecYE translocon from a bacterium, Thermus thermophilus. We determined the SecA-SecY contact residues and found that SecA and SecY interact with each other in at least two different modes. We proposed a new mechanism, by which SecA converts the energy stored in ATP into the movement of secretory precursor protein. We also carried out nearest neighbor analyses of SecYEG translocon subunits as well as analyses of the mode of involvement of the translocon in the formation of correctly folded membrane proteins. We found a new regulatory mechanism, in which an arrest sequence of SecM interacts with the exit tunnel of the ribosome, thereby regulating translation and folding of SecA. We have characterized the quality control system in the E. coli plasma membrane, in which FtsH, HtpX and associated factors play important roles. Our biochemical and biological characterization of membrane-integrated proteases has extended into two enzymes, RseP and G1pG, which catalyze intramembrane proteolysis. Finally, we have revealed the reaction mechanism and underlying structural bases of the DsbA-DsbB-uniquinone machinery that generates protein disulfide bonds to be introduced into client proteins.