Synthesis and assembly of H+-ATPase complex by isolated "rough" thylakoids

The synthesis and assembly of chloroplast H+-ATPase complex were studied by analyzing the incorporation of [35S]methionine into the constituent subunits with isolated intact chloroplasts and with thylakoid membranes that had been prepared from the chloroplasts so that they would retain ribosomes. The complex was isolated from thylakoids after labeling and identified by immunoprecipitation with an antiserum specific to CF1. The mechanism for the assembly of the complex was demonstrated to be active in the isolated chloroplasts by the following observations: the plastid genome-regulated subunits (alpha, beta, epsilon, I, and III) were labeled by in organello translation and recovered with the complex, and three other subunits (gamma, delta, and II) were labeled when intact chloroplasts were incubated with translation products from polyadenylated RNA. The two largest subunits, alpha and beta, were translated on thylakoid-bound ribosomes when the thylakoid membranes were incubated with soluble factors from Escherichia coli. They were recovered with the H+-ATPase complex, suggesting that they are translated on the bound ribosomes in the chloroplast, and that the isolated membranes retain the ability to assemble a complete complex. Provided that these observations are the result of de novo assembly of the complex, the imported and processed nuclear-coded subunits are presumed to be pooled not in stroma but on the membrane.