The Fate of Membrane-bound Ribosomes Following the Termination of Protein Synthesis

Abstract

Contemporary models for protein translocation in the mammalian endoplasmic reticulum (ER) identify the termination of protein synthesis as the signal for ribosome release from the ER membrane. We have utilized morphometric and biochemical methods to assess directly the fate of membrane-bound ribosomes following the termination of protein synthesis. In these studies, tissue culture cells were treated with cycloheximide to inhibit elongation, with pactamycin to inhibit initiation, or with puromycin to induce premature chain termination, and ribosome-membrane interactions were subsequently analyzed. It was found that following the termination of protein synthesis, the majority of ribosomal particles remained membrane-associated. Analysis of the subunit structure of the membrane-bound ribosomal particles remaining after termination was conducted by negative stain electron microscopy and sucrose gradient sedimentation. By both methods of analysis, the termination of protein synthesis on membrane-bound ribosomes was accompanied by the release of small ribosomal subunits from the ER membrane; the majority of the large subunits remained membrane-bound. On the basis of these results, we propose that large ribosomal subunit release from the ER membrane is regulated independently of protein translocation.