Abstract
The transport of large preproteins across the Escherichia coli plasma membrane is catalyzed by preprotein translocase, comprised of the peripherally bound SecA subunit and an integrally bound heterotrimeric domain consisting of the SecY, SecE, and SecG subunits. We have now placed the secY, secE, and secG genes under the control of an arabinose-inducible promoter on a multicopy plasmid. Upon induction, all three of the proteins are strongly overexpressed and recovered in the plasma membrane fraction. These membranes show a strong enhancement of 1) translocation ATPase activity, 2) preprotein translocation, 3) capacity for SecA binding, and 4) formation of the membrane-inserted form of SecA. These data establish that SecY, SecE, and SecG constitute the integral membrane domain of preprotein translocase.
Highlights
From the :j:Departr:zent of Biocher:zistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844 and the §Department of BIOlogy, Unioersity of Crete, 71110 lraklio, Crete, Greece
The transport of large preproteins across the Escherichia coli plasma membrane is catalyzed by preprotein translocase, comprised of the peripherally bound SecA subunit and an integrally bound heterotrimeric domain consisting of the SecY, SecE, and SecG subunits
We report that inner membrane vesicles prepared from cells with the secY, secE, and secG genes under the ara regulon on a multicopy plasmid are highly enriched in each of the SecYEG subunits
Summary
Translocation has been reproduced with all-purified components in a reconstituted reaction with proteoliposornes bearing SecYEG, SecA, proOmpA (the precursor of outer membrane protein A), a tiILH+ (established by the incorporation of bacteriorhodopsin in the liposomal membrane) and ATP (Brundage et al, 1990) In this reaction, translocase can support many turnovers of substrate, and the initial rates of translocation per functional translocase complex are similar to those seen with inner membrane vesicles (Bassilana and Wickner, 1993), Despite extensive biochemical, genetic, and physiological characterization of the translocation reaction, it has remained possible that there are additional subunits of the translocase enzyme. We report that inner membrane vesicles prepared from cells with the secY, secE, and secG genes under the ara regulon on a multicopy plasmid are highly enriched in each of the SecYEG subunits Both catalytic and stoichiometric measurements show a significant increase in translocation activity and sites
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