A cloned cpDNA fragment containing a portion of the gene for the 32 -36 kDa thylakoid protein of Chlamydomonas (polypeptide D-l) was isolated. Hybridization probing of RNA from soluble and membrane fractions of Chfamydomonas showed that the mRNA for D-l is bound to thylakoid membranes.Run-off translation of thylakoid-bound polysomes (rough thylakoids) with [35S]-methionine yields polypeptide D-l as the major product. Peptide mapping with S. aureus V-8 protease of D-l synthesized (1) in vivo, (2) in vitro by rough thylakoids and (3) in the reticulocyte lysate directed by non-polyadenylated RNA showed that D-l is synthesized as a precursor in the reticulocyte lysate but as the mature polypeptide by rough thylakoids. ChlamydomonaspsbA gene DNA-RNA hybridization Rough thylakoid In vitro protein synthesis 32 kDa precursor

An open reading frame potentially encoding a protein of 1995 amino acids (orf1995) has been found in the chloroplast genome of the green alga Chlamydomonas reinhardtii. Besides having a short hydrophobic N-terminal domain with five putative transmembrane helices, the predicted orf1995 product is highly basic. orf1995 might be a homologue of the ycf1 gene in land plants, whose function has not yet been determined. Mutants of C. reinhardtii transformed with a disruption of orf1995 remain heteroplasmic for the wild-type and disrupted alleles of this gene, indicating that the orf1995 product is essential for cell survival.

The amount of chloroplast ribosomal RNAs of Chlamydomonas reinhardtii which sediment at 15,000 g is increased when cells are treated with chloramphenicol . Preparations of chloroplast membranes from chloramphenicol-treated cells contain more chloroplast ribosomal RNAs than preparations from untreated cells . The membranes from treated cells also contain more ribosome-like particles, some of which appear in polysome-like arrangements . About 50% of chloroplast ribosomes are released from membranes in vitro as subunits by 1 mM puromycin in 500 mM KC1 . A portion of chloroplast ribosomal subunits is released by 500 mM KCl alone, a portion by 1 mM puromycin alone, and a portion by 1 mM puromycin in 500 mM KCl . Ribosomes are not released from isolated membranes by treatment with ribonuclease . Membranes in chloroplasts of chloramphenicol-treated cells show many ribosomes associated with membranes, some of which are present in polysome-like arrangements . This type of organization is less frequent in chloroplasts of untreated cells . Streptogramin, an inhibitor of initiation, prevents chloramphenicol from acting to permit isolation of membrane-bound ribosomes .

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