Reconstitution of CF , - depleted thylakoid membranes with complete and fragmented chloroplast ATPase The role of the 6 subunit for proton conduction through CFo

Chloroplast ATPase (CF,) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion-exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF, lacking the 6 & E subunits: CF1(-6) and CFl(-E). Washing Mono-Q-bound CFl with alcohol-containing buffers followed by elution without alcohol produced the p subunit and in separate peaks CF1( 6) and CFI( E ) . Elution from Mono Q in the presence of tenside yielded a p6 fragment, CF1(-6) and CF1(-&). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these ‘EDTA vesicles’ was obtained by addition of CF1 and its fragments. CF1, CFl( -6) and CF1(-6c) were active with crossreactivity between spinach, pea and maize. &containing CF1 always reconstituted higher activities than &deficient CF1. The p6 fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while j and DCCD-inhibited CF1(-6) were not. These results support the notion that subunit 6 can function as a stopcock to the CFo proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 307830821.