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Starting from isolated chloroplasts of the Chlamydomonas reinhardii cw 15 mutant, several mRNA-containing chloroplast subfractions, i.e. thylakoid-bound polysomes, detached polysomes or isolated RNA, were prepared and incubated in homologous and heterologous translation systems. In the reticulocyte lysate these fractions gave rise to strikingly different product patterns. A most prominent difference concerned the in-vivo rapidly labelled 32,000-dalton thylakoid polypeptide. Neither this membrane protein nor its 34,000-dalton precursor was formed when membrane-containing or free polysomes were translated, while the 34,000-dalton precursor was a main product of the RNA isolated from the same membranes. The influence of thylakoid membranes during translation was also observed in homologous translation systems with lysed chloroplasts supplemented with ATP. Membrane and soluble fractions, when translated separately, yielded product patterns which differed from each other, although the RNAs extracted from the respective fractions gave the same product patterns when translated in reticulocyte lysate; the latter included a soluble protein, the large subunit of ribulose-1,5-bisphosphate carboxylase, and a membrane protein, the 34,000-dalton precursor of the 32,000-dalton membrane protein, as major labelled translation products. These results point to a regulatory role of thylakoid membranes in the expression of chloroplast mRNA and argue against compartmentation of the chloroplast mRNAs between the soluble and membrane fractions.

A membrane fraction enriched in chloroplast envelopes was isolated from the alga Chlamydomonas reinhardii. The envelope preparations contained the envelope marker enzyme, galactosyltransferase, with a specific activity about 26- and 37-fold greater than that of the intact chloroplasts and of the starting cells, respectively. Both the envelope fraction and intact chloroplasts incorporated labelled galactose mostly into digalactosyldiglyceride (DGDG), with little incorporation into monogalactosyldiglyceride (MGDG), in marked contrast to what had been observed in higher plants. The envelope preparation was characterized in terms of its composition in polar lipids and polypeptides, and certain features were found in common with the corresponding membranes of higher plants. The ratio of galactolipids MGDG/DGDG was typically lower in the envelopes, compared to the thylakoid fraction. Analysis of the proteins by electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed a polypeptide pattern that was distinct from those of the thylakoids or of the soluble fraction of the chloroplasts. The major component of the envelope fraction was a 29-hilodalton polypeptide, similar to the main envelope polypeptide found in higher plants and believed to be the phosphate translocator.

Chloroplasts from the cell wall mutant cw-15-2 of Chlamydomonas reinhardii were isolated by disruption of the cells in the Yeda press and fractionation through step gradients of Percoll. The resulting chloroplast fraction contained 80–85% intact chloroplasts. Electron micrographs of thin sections of the chloroplast fraction showed some cytoplasmic impurities, although almost no cytoplasmic ribosomes were detected by analysis of the ribosomal subunits. The isolated chloroplasts are active in photosynthetic O 2 -evolution and CO 2 -fixation, with the highest rates obtained in the presence of ATP. The chloroplast fraction also showed high rates of light-dependent in organello protein synthesis, with labelling of discrete chloroplast proteins known to be synthesized in the chloroplasts.

Chloroplasts which have been isolated from the cell wall mutant cw15-2 of Chlamydomonas reinhardii are active in light-dependent, in organello protein synthesis. Certain characteristics of protein synthesis in this system were tested, such as time course of amino acid incorporation, sensitivities to Mg2+ as well as to specific inhibitors, and found to be typical of chloroplasts isolated from higher plants. The major products made were identified as the large subunit of ribulose-1,5-biphosphate carboxylase and the 32-kDa rapidly labelled membrane protein.

Studies on protein synthesis by isolated chloroplasts have contributed a great deal to our present knowledge of the biogenesis of chloroplast proteins. So far, the available information has been obtained with only a few higher plants and algae. In Chlamydomonas, studies on chloroplast protein synthesis have been carried out indirectly: in vivo by pulse-labelling the cells in the presence of specific inhibitors (Chua, Gillham 1977), or in vitro using polyribosomes, free or thylakoid-bound, or chloroplast DNA (for review, see Ellis 1981).

Chloroplast polysomes that were originally bound to thylakoid membranes were isolated from the cell wall mutant CW-15 from Chlamydomonas reinhardii . Polysomes were isolated from synchronously gorwn cells harvested in the middle of the third light period, when the ratio of chloroplast to cytoplasmic polysomes was maximal. Thylakoid membranes were isolated from a chloroplast fraction and polysomes were released by Triton X-100. Analyses of subunits on sucrose gradients showed that the polysomes consisted predominantly of the 70 S-type ribosomes. The detached polysomes as well as polysomes still bound to the thylakoid membrane were active in in vitro protein synthesis when supplemented with Escherichia coli -soluble factors. The in vitro activity was inhibited by chloramphenicol and aurintricarboxylic acid, but not by cycloheximide.