Cross-linking of chloroplast F0F1-ATPase subunit epsilon to gamma without effect on activity epsilon and gamma are parts of the rotor

Abstract

Cys residues were directed into positions 17, 28, 41 and 85 of a Cys6-->Ser mutant of subunit epsilon of spinach chloroplast F0F1 ATP synthase. Wild-type and engineered epsilon were expressed in Escherichia coli, purified in the presence of urea, refolded and reassembled with spinach chloroplast F-1 lacking the epsilon subunit [F-1(-epsilon)]. Cys-containing epsilon variants were modified with a sulfhydryl-reactive photolabile cross linker. Photocross-linking of epsilon to F-1(-epsilon) yielded the same SDS gel pattern of cross-link products independent of the presence of absence of Mg2+ . ADP, phosphate and Mg2+ . ATP. epsilon (wild type) [Ser6,Cys28]epsilon and [Ser6,Cys41]epsilon were cross-linked with subunit gamma. With chloroplast F0F1 the same cross-link pattern was obtained, except for one extra cross-link, probably between [Ser6,Cys28]epsilon and F-0 subunit III. [Ser6, Cys17]epsilon and [Ser6,Cys85]epsilon did not produce cross-links. Cross-linking of epsilon, [Ser6,Cys28]epsilon, [Ser6,Cys41]epsilon to gamma in soluble chloroplast F-1 impaired the ability of epsilon to inhibit Ca2+-ATPase activity. The Mg2+-ATPase activity of soluble F-1 (measured in the presence of 30% MeOH) was not affected by cross-linking epsilon with gamma. Functional reconstitution of photophosphorylation in F-1-depleted thylakoids was observed with F-1 in which gamma was cross-linked to [Ser6,Cys28]epsilon or [Ser6,Cys41]epsilon but not with wild-type epsilon. In view of the intersubunit rotation of gamma relative to (alpha beta)(3), which is driven by ATP hydrolysis, gamma and epsilon would seem to act concertedly as parts of the `rotor' relative to the `stator' (alpha beta)(3).