Biosynthesis and assembly of the proton-translocating adenosine triphosphatase complex from chloroplasts

Abstract

The H(+)-translocating ATPase complex of chloroplasts consists of at least eight nonidentical subunits. Five of these (alpha, beta, gamma, delta, and epsilon subunits) collectively constitute the globular extramembranous CF(1) portion of the complex. The remaining three subunits (I-III) represent the membrane-embedded portion. Biosynthesis and assembly of these subunits were studied by pulse-labeling isolated spinach chloroplasts in the presence of cycloheximide or chloramphenicol and by translating total leaf RNA in a rabbit reticulocyte system. The labeled products were analyzed by immunoprecipitation with subunit-specific antisera or by isolating the entire H(+)-translocating ATPase complex in a nearly pure state. We found that chloroplasts synthesize the alpha, beta, gamma, and epsilon subunits of CF(1), the membrane-embedded subunit I, and probably also the membrane-embedded subunit III. The delta subunit (and probably also subunit II) are imported from the cytoplasm via larger precursor forms. After isolated chloroplasts are labeled in the presence of cycloheximide, the chloroplast-made H(+)-ATPase subunits are assembled into a complex that is indistinguishable from the authentic H(+)-ATPase complex. This assembly indicates that isolated chloroplasts contain excess pools of the cytoplasmically made subunits.