Biochemical analysis of the biogenesis and function of the Escherichia coli export factor SecY

Open Access
Publisher Website
Google Scholar
Abstract
SecY is an integral plasma‐membrane protein of Escherichia coli which is essential for the export of periplasmic and outer‐membrane proteins containing cleavable signal sequences. We have synthesized SecY in vitro using an E. coli transcription/translation system. In the absence of membranes, SecY remained largely soluble but cosedimented on sucrose gradients with the membrane fraction when inside‐out plasma‐membrane vesicles (INV) had been added cotranslationally. Membrane association of SecY was unaffected if the endogenous SecY of the INV had been inactivated by either antibodies, a mutation or trypsin treatment. In contrast, inactivation of the INV SecY interfered with membrane targeting and, consequently, the processing of precursors to β‐lactamase and λ receptor. When SecY‐deprived INV were, however, first functionally reconstituted with in‐vitro‐ synthesized SecY, targeting and translocation of λ receptor were partially resorted. Thus, the assembly of SecY into INV in vitro leads to an active enzyme. In addition, we show that the prlA4 allele of the secY gene suppresses signal‐sequence mutatios of the λ receptor in vitro. Collectively, our results demonstrate that SecY, while funcitoning as a membrane‐located receptor for precursors of exported proteins, appears to be virtually independent of pre‐existing SecY for its own membrane integration.