Sedimentation behavior of chloroplast ribosomes from Chlamydomonas reinhardtii

Abstract

The identity of peaks generated by chloroplast ribosomes of Chlamydomonas reinhardtii were determined by zone velocity sedimentation on sucrose density gradients, and analysis of distribution of ribosomal RNAs in the gradients. The sedimentation coefficient of the principal peak was 66–70 S (usually 69 S), in good agreement with previously reported values for chloroplast ribosomes of C. reinhardtii, and other organisms. The fast sedimenting side of the 69 S peak contained an excess of chloroplast large subunit. When ribosome dissociation was prevented by sedimentation at low velocity, by aldehyde fixation, or by the presence of nascent polypeptide chains, the principal peak had a sedimentation coefficient of about 75 S. Thus the 69 S peak was an artifact caused by dissociation during centrifugation. Peaks that contained chloroplast ribosomal RNAs were also observed at ‘60 S’ and ‘45 S’ when chloroplast ribosomes were centrifuged unfixed at high velocity. The amounts of ‘60 S’ and ‘45 S’ components were decreased by centrifugation at low speed, or fixation, but sedimentation coefficients remained unchanged. The ‘60 S’, and ‘45 S’ components were identified as large, and small subunits of chloroplast ribosomes, respectively. The artifacts produced by centrifugation of chloroplast ribosomes, are similar to the artifacts produced by centrifuging ribosomes of Escherichia coli. Similar explanations appear to apply to both. We concluded that the 69 S chloroplast ribosome peak occurs because of dissociation of ‘tight’ couples, and incomplete separation of subunits. Subunit peaks (60 S and 45 S) arise from free subunits, and/or from dissociation of ‘loose’ couples.