Reconstitution of CF1-depleted thylakoid membranes with complete and fragmented chloroplast ATPase.

Chloroplast ATPase (CF1) 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 CF1 lacking the &delta ; & ε subunits: CF1(-&delta ;) and CF1(-&ε;). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the β subunit and in separate peaks CF1(-&delta ;) and CF1(-ε). Elution from Mono Q in the presence of tenside yielded a β &delta ; fragment, CF1(-&delta ;) and CF1(-&delta ; ε). Chloroplasts were CF1-depleted by EDTA extraction. Reconstitution of photophosphorylation in these 'EDTA vesicles' was obtained by addition of CF1 and its fragments. CF1, CF1(-&delta ;) and CF1(-&delta ; ε) were active with cross-reactivity between spinach, pea and maize. &delta ;-containing CF1 always reconstituted higher activities than &delta ;-deficient CF1. The β &delta ; fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while β and DCCD-inhibited CF1(-&delta ;) were not. These results support the notion that subunit &delta ; can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078-3082]. [ABSTRACT FROM AUTHOR]