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

Subunit interactions among the chloroplast ATP synthase subunits were studi ed using the yeast two-hybrid system. Various pairwise combinations of gene s encoding alpha, beta, gamma, delta and epsilon subunits of Spinach ATP sy nthase fused to the binding domain or activation domain of GAL4 DNA were in troduced into yeast and then expression of a reporter gene encoding beta-ga lactosidase was detected. Of all the combinations, that of gamma and epsilo n subunit genes showed the highest level of reporter gene expression, while those of alpha and beta, alpha and epsilon, beta and epsilon and beta and delta induced stable and significant reporter gene expression. The combinat ion of delta and epsilon as well as that of delta and gamma induced weak an d unstable reporter gene expression. However, combinations of alpha and gam ma, beta and gamma and alpha and delta did not induce reporter gene express ion. 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.