In vitro translocation of periplasmic and outer membrane proteins into inverted plasma membrane vesicles from Escherichia coli was completely prevented by the H+‐ATPase inhibitor N,N′‐dicyclohexylcarbodiimide (DCCD). DCCD was inhibitory to both co‐ and post‐translational translocations, suggesting an involvement of the H+‐translocating F1F0‐ATPase in either mode of transport. This was verified by (i) the dependence of efficient co‐translational translocation upon a low salt, i.e. F1‐containing extract from membrane vesicles; (ii) the co‐purification of the translocation activity present in this extract and F1‐ATPase; (iii) the inability of either vesicles or their low‐salt extract, derived from F1F0‐ATPase‐lacking mutant strains, to support translocation; and (iv) the greatly diminished extent of ATP‐dependent, post‐translational translocation into F1‐deprived vesicles. Membranes devoid of F1 did show, however, residual translocation activity that was also found to be inhibitable by DCCD. These results suggest a dual target for DCCD in bacterial protein export, one being the H+‐ATPase and the other an as yet unidentified translocation factor.
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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