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.

The Permeability of the Endoplasmic Reticulum Is Dynamically Coupled to Protein Synthesis

“…Also, the increase in P basal produced by both PUR and PAC was about 70% of the ⌬P PUR measured in control cells (Fig. 4, A and B, where ⌬P PUR reflects the size of the translationally active pool in control cells), which was remarkably consistent with the previous report of a persistent binding of Ϸ65% of 60 S subunits after the release of nascent proteins (5).…”

“…The increased permeability to 4-M␣G was consistent with a previous report that RBTs incorporated into planar bilayers and opened by puromycin are permeable to ions (3), and our results were also supported by a recent report that puromycin can release calcium from the RER (4). The permeation of empty RBTs is especially significant in the context of recent studies by Nicchitta and colleagues (5,6), who reported that approximately two-thirds of the 60 S subunits remain bound to translocons after the normal completion of protein translation, thereby constituting a large pool of persistent, translationally inactive RBT complexes. Although permeation of these empty RBTs could have very important consequences for RER-dependent signaling and the maintenance of essential gradients across the RER membrane, this pathway has received relatively little attention.…”

“…2B). The similarity between CHO-S and HepG2 cells is especially notable, because Seiser and Nicchitta (5) reported that approximately twothirds of the ribosomes remain bound to the RER membrane in HepG2 cells following either the premature termination of translation by PUR or the normal completion of translation with pactamycin. We conclude from these similarities that the mechanisms that regulate the permeation of RBTs are probably also similar among these cell lines and that the high P basal and the ⌬P PUR are not unusual features of CHO-S cells.…”

Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

“…3, D and E), indicating that agonist-stimulated iNOS activation is independent of changes in the levels of iNOS protein expression. As a further support to this conclusion, we also obtained data that preincubation with a protein synthesis inhibitor puromycin under conditions shown previously to abolish protein translation (22,23) did not affect the thrombin-induced NO production in wild-type or eNOS knock-out platelets (data not shown). Consistent with these results, we have also obtained data that there is no significant de novo synthesis of Bcl-3, a protein shown to be synthesized after prolonged platelet activation, after 5 min of agonist stimulation of mouse platelets under the same experimental conditions that iNOSdependent NO synthesis was demonstrated (data not shown).…”

The selective recruitment of mRNA to the ER and an increase in initiation are important for glucose-stimulated proinsulin synthesis in pancreatic β-cells

“…This technique has been previously used to successfully fractionate cytosolic mRNAs from ER-bound mRNAs [23].…”