Evidence for Synthesis of Proteins of Chloroplastic Ribosomes Ox Cytoplasmic Ri- Bosomes*

The biosynthesis of proteins of chloroplastic (67 S) and cytoplasmic (83 S) ribosomes of Chlamydomonas reinhardi was studied in viva using both wild type and the arginine auxotroph, arg-1. The incorporation of amino acids into nascent protein (chaseable label) and ribosomal structural protein (nonchaseable label) was examined. When cells of tug-1 were incubated with excess radioactive arginine, 83 S ribosomes were clearly labeled at 0.5 to 2.0 min. But, 67 S ribosomes were not, even though chloroplasts were carrying out protein synthesis. At these times much of the label in 83 S ribosomes was in nascent protein, but no labeled protein was found with 67 S ribosomes. When cells were pulse-labeled for 2 min and incubation continued, label was found in nascent protein of 83 S ribosomes at 2 and 4 min, but label was found only in structural protein at later times. In contrast, 67 S ribosomes did not become labeled until 6 min, and at no time from 2 to 20 min was label in nascent protein. When pulse-labeled cells were chased prior to 4 min, label in 83 S ribosomes decreased while label in 67 S ribosomes increased. At 6 min, chase had no effect on label in 83 S ribosomes, but increased label in 67 S ribosomes. The results could be explained if there was a delay in labeled amino acid reaching the chloroplast, or if 67 S ribosomes lost nascent protein during isolation. The results also indicated that labeled protein in 67 S ribosomes was not made on 67 S ribosomes, but appeared instead to have come from a pool of free labeled proteins whose ultimate source was nascent protein on 83 S ribosomes. When cells of arg-1 were labeled with radioactive arginine for 1 hour, both 67 S and 83 S ribosomes were clearly labeled, and were of equal specific radioactivities. Radioactivity was in structural protein. Similar results were obtained using wild type and radioactive leucine. Chloramphenicol was without detectable effect on labeling of structural protein of 67 S ribosomes, whether labeling was accomplished by incubating cells with radioactive amino acid for 2 min and chasing with unlabeled amino acid, or by incubating cells with radioactive amino acid for 60 min. Chloramphenicol, however,