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

TLDR: 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, which indicates that isolatedchloroplasts contain excess pools of the cytoplasmically made subunits.

Abstract: The H+-translocating ATPase complex of chloroplasts consists of at least eight nonidentical subunits. Five of these (α, β, γ, δ, and e subunits) collectively constitute the globular extramembranous CF1 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 α, β, γ, and e subunits of CF1, the membrane-embedded subunit I, and probably also the membrane-embedded subunit III. The δ 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.