The long-term objective is understanding the mechanism by which proteins cross through membranes. The proposed research is a biochemical characterization of the energy requirements for protein transport across the Escherichia coli inner membrane. An established, in vitro, bacterial protein export assay will be used to measure the effects of both ATP and a transmembrane electrochemical potential on protein export. The in vitro assay allows these energy sources to be carefully controlled and measured during the movement of proteins across inner membrane vesicles. Previous results have shown that both ATP and an electrochemical potential are necessary for protein export in vitro. By sequentially adding ATP and a transmembrane electrochemical potential to the in vitro assay, the order and timing of the energy requirements can be understood with respect to the individual molecular steps that occur during this complex reaction. The requirement for ATP, independent of the electrochemical potential, suggests that ATP hydrolysis may be necessary for protein export. Preliminary evidence indicates that during protein export, the terminal phosphate residue of ATP is transferred to a cellular component with an apparent size of about 30,000 daltons. The effects of inhibitors of protein export on this phosphate group transfer will be measured. A correlation between the extent of inhibition of protein export and phosphate transfer would further define the role of ATP in protein export. The phosphate label may allow a means of isolating this potentially important component in protein export.
