Ribosome Substructure in Intact Mouse Liver Cells

Florendo, Noel T.

doi:10.1083/jcb.41.1.335

Cited by 30 publications

(9 citation statements)

References 16 publications

(9 reference statements)

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“…Thus, if isolated ER microsomes were extracted with increasing concentrations of EDTA, the small ribosomal subunit was preferentially released from the membrane, whereas the large ribosomal subunit remained membrane-bound (7,8). Confirmation of the nature of ribosome binding to the ER was later obtained by direct ultrastructural analysis of intact cells, where it was observed that the large ribosomal subunit contains the site of membrane attachment (10).…”

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confidence: 83%

“…In the pioneering studies on ribosome-membrane interactions in the ER, it was observed that ribosomes bind asymmetrically, with the binding interaction being mediated entirely through the large subunits (7)(8)(9)(10). Thus, if isolated ER microsomes were extracted with increasing concentrations of EDTA, the small ribosomal subunit was preferentially released from the membrane, whereas the large ribosomal subunit remained membrane-bound (7,8).…”

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confidence: 99%

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

Seiser

Nicchitta

2000

Journal of Biological Chemistry

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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.

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confidence: 83%

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confidence: 99%

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

Seiser

Nicchitta

2000

Journal of Biological Chemistry

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“…Nonomura et al . Manuscript in preparation), while in positively stained ribosomes examined in sections of material fixed in Os04 it varies between X150 and 200 A (25) .…”

Section: Liver Free Ribosomesmentioning

confidence: 98%

Controlled Proteolysis of Nascent Polypeptides in Rat Liver Cell Fractions

Sabatini

Blobel

1970

The Journal of Cell Biology

310

121

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Free ribosomes containing nascent polypeptide chains labeled in vitro were submitted to proteolysis at 0° by a mixture of trypsin and chymotrypsin. Sucrose gradient analysis showed that polysome patterns are retained even after 24 hr of proteolysis in the cold, while messenger RNA-free ribosomes (generated progressively during in vitro incorporation) are, within 2 hr, completely dissociated into subunits by trypsin. Although ribosomes and subunits are not extensively degraded into smaller fragments during low temperature proteolysis, changes in the acrylamide gel electrophoresis pattern showed that most ribosomal proteins are accessible to and are partially degraded by the proteases. Ribosome-bound nascent polypeptides are partially resistant to proteolysis at 0°, although they are totally digested at 37° or when the ribosomal subunit structure is disrupted by other means. Radioactivity incorporated into nascent chains during incubation times shorter than 3 min was mostly resistant to digestion at 0°. A larger fraction of the initial radioactivity became degraded in ribosomes which incorporated for longer times. In these ribosomes, the amount of radioactivity which was resistant to proteolysis was constant and independent of the initial value, which reflects the labeled length of the nascent chains. These results suggest that the growing end of the nascent polypeptide is resistant to digestion and is protected from proteolytic attack by the ribosomal structure. A pulse and chase experiment confirmed this suggestion, showing that the protected segment is located at the carboxy-terminal end of the nascent chain. The protected segment was contained in the large ribosomal subunit and had a length of ∼39 amino acid residues, as estimated by chromatography on Sephadex G-50.

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“…It is known that ribosomes interact with microsomal membranes via the large (60S) subumt (16,17) and that the nascent polypeptide chain, which grows within a protected region in this subunit (18,19), enters into close relationship with the membrane immediately upon emerging from the ribosome (20) Nevertheless, both in vivo and in vitro studies with puromycin (14,21,22) have failed to demonstrate a role for the nascent chain in binding the ribosomes to the membranes. Similarly, there is no indication that in rat liver RM ribosomemembrane interaction is mediated by messenger RNA (21, also extensive unpublished studies by T. Morimoto and D. Sabatini involving RNase digestions), although recent publications have suggested such a binding mechanism exists in tissue cultme ceils (23,24).…”

Section: Introductionmentioning

confidence: 99%

Ribosome-Membrane Interaction

Adelman

Sabatini

Blobel

1973

The Journal of Cell Biology

316

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A B S T R A C TIn a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KC1, 5 mM MgCl~, 50 m2~ Tris.HC1, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [SH]puromycin into hot acid-insoluble material and from the release of [SH]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25-100 rnM) as at high (500-1000 raM) KC1 concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KC1 concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction ( N I 5 % ) of the bound ribosomes can only be released from membranes by exposure of R M to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.

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Ribosome Substructure in Intact Mouse Liver Cells

Cited by 30 publications

References 16 publications

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

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

Controlled Proteolysis of Nascent Polypeptides in Rat Liver Cell Fractions

Ribosome-Membrane Interaction

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