title: DCCD inhibits protein translocation into plasma membrane vesicles from Escherichia coli at two different steps.
abstract: 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.
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D002234:Carbodiimides / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D011499:Protein Processing, Post-Translational; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002234:Carbodiimides; D004024:Dicyclohexylcarbodiimide; D006180:Proton-Translocating ATPases