Subunit interactions of chloroplast ATP synthase from higher plant

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. The coding sequences of a, b, g, d and e subunit of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed in the yeast strain SFY526 by various pairwise combinations, and the subunit interactions were analyzed by detecting the expression activity of a reporter gene encoding b -galactosidase. The results showed that specific and strong interactions between g and e, a and b, a and e, b and e, b and d subunits, and weak and transient interactions between d and e, d and g subunits occurred in the yeast cell in the two-hybrid assay. We also observed that the inhibition of e subunit on Ca2+-ATPase was related to its affinity with other parts of CF1. Ten truncation mutants of e subunit that sequential lacking 1 to 5 amino acids from the N terminus and 6 to 10 amino acids from the C terminus and four site-directed mutants, e T42C, e T42R, e T42I and e T42P were generated by PCR and overexpressed in E. Coli. The expressed e proteins were purified and recombined with soluble and membrane-bound CF1, the ATP synthesis and hydrolysis activity of ATP synthase, the proton conductance of membrane and the direct interactions of e with other subunits were examined. All these results suggested that the interactions between the e subunit and other subunits of CF1 played an important role in regulating its inhibitory function to chloroplast ATP synthase.