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 p-lactamase and , I receptor. When SecYdeprived INV were, however, first functionally reconstituted with in-vitro-synthesized SecY, targeting and translocation of the , I receptor were partially restored. Thus, the assembly of SecY into INV in vitro leads to an active enzyme. In addition, we show that the prlA4 allele of the sec Y gene suppresses signal-sequence mutations of the , I receptor in vitro. Collectively, our results demonstrate that SecY, while functioning as a membrane-located receptor for precursors of exported proteins, appears to be virtually independent of pre-existing SecY for its own membrane integration.
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