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

Abstract

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. and .epsilon. subunits: CF1(-.delta.) and CF1(-.epsilon.). Washing Mono-Q-bound CF1 with alcohol-containing buffers followed by elution without alcohol produced the .beta. subunit and in separate peaks CF1(-.delta.) and CF1(-.epsilon.). Elution from Mono Q in the presence of tenside yielded a .beta..delta. fragment, CF1(-.delta.) and CF1(-.delta..epsilon.). 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..epsilon.) were active with cross-reactivity between spinach, pea and maize. .delta.-containing CF1 always reconstituted higher activities than .delta.-deficient CF1. The .beta..delta. fragment and dicyclohexylcarbodiimide (DCCD)-inhibited CF1 also were reconstitutively active while .beta. 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].