Cross‐reconstitution studies with polypeptides of Escherichia coli and bovine heart mitochondrial FoF1 ATP synthase

Abstract

To characterize the role of supernumerary subunits of the mammalian FoF1 ATP synthase, crossreconstitution of mitochondrial and bacterial FoF1 complexes has been carried out. Escherichia coli F1 (EcF1) can be reconstituted with F1‐stripped everted membranes of E. coli (UPEc) and of bovine heart mitochondria (USMP). Bovine heart mitochondrial F1 (BHF1) can also be reconstituted with both membranes. Both EcF1 and BHF1, when reconstituted with UPEc, exhibited oligomycin‐insensitive ATP‐hydrolase activity. Subunits of the mammalian Fo, in particular FoI‐PVP protein, F6 and oligomycin‐sensitivity‐conferring protein (OSCP) conferred oligomycin sensitivity to the catalytic activity of EcF1 or BHF1 reconstituted with UPEc. Reaction of N, N′‐dicyclohexylcarbodiimide and development of inhibition of passive H+ conduction was, in UPEc, considerably slower and exhibited a lower apparent affinity than in USMP. The ATP hydrolase activity of UPEc+EcF1 or UPEc+BHF1 was, also, less sensitive to inhibition by N, N′‐dicyclohexylcarbodiimide than USMP+EcF1 or USMP+BHF1. Addition of mitochondrial FoI‐PVP to UPEc enhanced the sensitivity of H+ conduction to oligomycin. FoI‐PVP and OSCP added to UPEc, promoted inhibition by N, N′‐dicyclohexylcarbodiimide of passive H+ conduction and increased its binding affinity to subunit c of E. coli Fo. The presence of FoI‐PVP and OSCP also promoted inhibition by N, N′‐dicyclohexylcarbodiimide of the ATP‐hydrolase activity of EcF1 or BHF1 reconstituted with UPEc. Copyright © 1994, Wiley Blackwell. All rights reserved