Using yeast two-hybrid system to detect interactions of ATP synthase subunits fromSpinacia oleracea

Abstract

Subunit interactions among the chloroplast ATP synthase subunits were studied using the yeast two-hybrid system. Various pairwise combinations of genes encoding alpha, beta, gamma, delta and epsilon subunits of Spinach ATP synthase fused to the binding domain or activation domain of GAL4 DNA were introduced into yeast and then expression of a reporter gene encoding beta-galactosidase was detected. Of all the combinations, that of gamma and epsilon subunit genes showed the highest level of reporter gene expression, while those of alpha and beta, a and epsilon, beta and epsilon and beta and delta induced stable and significant reporter gene expression. The combination of delta and epsilon as well as that of delta and gamma induced weak and unstable reporter gene expression. However, combinations of alpha and gamma, beta and gamma and alpha and delta did not induce reporter gene expression. These results suggested that specific and strong interactions between gamma and epsilon, alpha and beta, alpha and epsilon, beta and epsilon and beta and delta subunits, and weak and transient interactions between delta and epsilon and delta and gamma subunits occurred in the yeast cell in the two-hybrid system. These results give a new look into the structural change of ATP synthase during catalysis.