Membrane vesicles containing overproduced SecY and SecE exhibit high translocation ATPase activity and countermovement of protons in a SecA- and presecretory protein-dependent manner.

Abstract

Everted membrane vesicles were prepared from Escherichia coli cells containing either overproduced amounts (OP-membrane vesicles) or normal amounts (normal membrane vesicles) of SecY and SecE, both of which are essential components of the protein translocation apparatus. The rates of translocation of pro-OmpA were similar in the two types of membrane vesicles, whereas translocation ATPase activity, which requires SecA, a precursor protein (pro-OmpA), and membrane vesicles, was appreciably higher with OP-membrane vesicles than with normal membrane vesicles. Since ATP hydrolysis has been shown to take place at an earlier part of the translocation reaction, these results suggest that the overproduction of SecY and SecE enhanced the activity of the earlier process, but not the entire process, of the translocation reaction. The addition of pro-OmpA in the presence of SecA caused the partial collapse of delta pH (inside acidic) generated on OP-membrane vesicles, suggesting that protons come out from the inside of the membrane vesicles in a pro-OmpA-dependent manner. The collapse of delta pH caused by pro-OmpA required SecA, ATP, and SecY and was not detected when normal membrane vesicles were used. These results indicate that the early event of protein translocation, which requires the functioning of SecA, SecY, and SecE, causes the countermovement of protons.