Biogenesis of Chloroplast Membranes: VI. COOPERATION BETWEEN CYTOPLASMIC AND CHLOROPLAST RIBOSOMES IN THE SYNTHESIS OF PHOTOSYNTHETIC LAMELLAR PROTEINS DURING THE GREENING PROCESS IN A MUTANT OF CHLAMYDOMONAS REINHARDI y-1

Abstract

Abstract Proteins from a membrane fraction enriched in chloroplast material obtained by density gradient centrifugation were analyzed by electrophoresis on acrylamide gels. About 15 to 20 bands could be resolved. The electrophoretic pattern of the proteins obtained from dark-grown cells, lacking the chloroplast lamellae, differed from that of light-grown photosynthetic cells. When dark-grown cells were exposed to light, a main protein peak designated as L protein appeared parallel to the increase in the chlorophyll content. Continuous labeling and pulse labeling of the proteins during the greening process, in the presence or absence of cycloheximide and chloramphenicol, indicated that the L protein fraction is synthesized de novo; its synthesis is inhibited by cycloheximide but not by the latter compound. Membranes synthesized in the presence of chloramphenicol have a very low photosynthetic activity although a normal amount of L protein is present in the cells. Removal of the inhibitor allows synthesis of new proteins and activation of the defective membranes. This process does not require synthesis of additional L protein or chlorophyll since it is not inhibited by cycloheximide and it can occur in the dark. Pulse labeling of proteins after transfer of greening cells from chloramphenicol to cycloheximide indicates progressive reduction in the labeling of the L protein peak and appearance of radioactivity in other protein peaks which are not labeled in the presence of chloramphenicol. Cycloheximide completely blocks the synthesis of chlorophyll and protein when added at any time during the greening process in a normal noninhibited system. However, prior incubation of the greening cells with chloramphenicol leading to accumulation of L protein protects the system against the complete inhibition exerted by cycloheximide. Based on these data it is suggested that in the process of synthesis and assembly of the photosynthetic membranes, proteins of cytoplasmic and chloroplast origin concur to form a functional membrane. The synthesis of these two types of protein is regulated by a feedback-like mechanism; its features include control of the level of the lamellar proteins of cytoplasmic origin by the level of chlorophyll and control of the level of proteins of chloroplast origin by that of the cytoplasmic proteins. In addition, the light-dependent chlorophyll synthesis is controlled by the level of the lamellar proteins of chloroplasts and cytoplasmic origin.