Synthesis of EF-Tu and distribution of its mRNA between stroma and thylakoids during the cell cycle of Chlamydomonas reinhardii

In Chlamydomonas reinhardii the elongation factor EF-Tu is encoded in the chloroplast DNA. We identified EF-Tu in the electrophoretic product pattern of chloroplast-made proteins and showed that this protein is only synthesized in the first half of the light period in synchronized cells. The newly synthesized EF-Tu contributed little to the almost invariable content of EF-Tu in chloroplasts during the light period of the cell cycle. However, increasing cell volume and the lack of EF-Tu synthesis in the second half of the light period led to a decrease in the concentration of EF-Tu in chloroplasts. At different times in the vegetative cell cycle, the RNA was extracted from whole chloroplasts and from free and thylakoid-bound chloroplast polysomes. The content of mRNA of EF-Tu in chloroplasts and the distribution between stroma and thylakoids were determined. During the light period, the content of the mRNA for EF-Tu varied in parallel to the rate of EF-Tu synthesis. However, in the dark, some mRNA was present even in the absence of EF-Tu synthesis. Most of the mRNA was bound to thylakoids during the whole cell cycle. This suggests that synthesis of EF-Tu is associated with thylakoid membranes.     

Herbicide-binding to thylakoid membranes of a DCMU-resistant mutant of Chlamydomonas reinhardii

The specific binding of DCMU and atrazine to the thylakoid membranes of a uniparentally inherited DCMU-resistant mutant dr-416 of Chlamydomonas reinhardii was measured. Whole cells of the mutant can tolerate a 15-fold concentration of DCMU as compared to the parent strain. The same tolerance is found for the photosystem II activity of isolated thylakoid membranes. The mutant is not resistant against atrazine. In equilibrium-binding studies with [¹⁴C]atrazine and unlabelled DCMU, the specific binding for atrazine was found to be identical in the mutant and in the parent strain. The competitive binding of DCMU is significantly weaker for membranes of the mutant than of the parent strain, the equilibrium dissociation constants being 2.0 × 10^?7 M and 3.8 × 10^?8 M, respectively.     

In-vitro translation of different mRNA-containing fractions of Chlamydomonas chloroplasts

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.Abbreviation SDS sodium dodecyl sulfate