Attachment of Ribosomes to Membranes During Polysome Formation in Mouse Sarcoma 180 Cells

Lee, Se Yong; Krsmanovic, V.; Brawerman, George

doi:10.1083/jcb.49.3.683

Cited by 63 publications

(21 citation statements)

References 8 publications

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“…In agreement with previous reports (Loeb et al, 1967;Talal & Kaltreider, 1968;Ragnotti et al, 1969;Tanaka et al, 1970;Lee et al, 1971;Venkatesan & Steele, 1972;Mishra et al, 1972), our results suggest first that the two classes of polyribosomes present within the liver cell, though morphologically distinct, are not functionally separate and secondly that the membrane-ribosome interaction is a dynamic relationship that shifts as the functional requirements of the cell demand.…”

Section: Vol 146supporting

confidence: 93%

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats

Ragnotti

Aletti

1975

Biochemical Journal

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1. The effect of phenobarbitone on the rate of protein synthesis and on the sedimentation patterns of various liver subcellular fractions containing ribosomes was studied in rats. 2. Phenobarbitone treatment increased the incorporation of [14C]leucine into protein by all preparations, provided they had not been subjected to preliminary treatment with Sephadex G-25. The phenobarbitone-induced effect on incorporation was associated with a gain in liver weight and a higher degree of polyribosomal aggregation. 3. Preparations that were treated with Sephadex G-25 incorporated more radioactivity into protein, but did not show the response to phenobarbitone treatment. 4. When the influence of starvation and phenobarbitone was studied separately on membrane-bound and membrane-free polyribosomes, it was shown that whereas both classes of polyribosomes were affected by starvation, apparently only the former class was susceptible to phenobarbitone stimulation of protein synthesis. 5. The decreased capacity for protein synthesis of polyribosomes from starved rats was independent of their association with the membranes of the endoplasmic reticulum, but resulted from polyribosomal disaggregation, from an intrinsic defect of the polyribosomes themselves and from changes in composition of the cell sap. 6. The results are discussed in relation to the problem ofthe control ofprotein biosynthesis and of the functional separation of membrane-bound and membrane-free polyribosomes.

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Section: Vol 146supporting

confidence: 93%

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats

Ragnotti

Aletti

1975

Biochemical Journal

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“…Previous evidence tends to suggest that polysome formation is accompanied by association of the mRNA with a large structure (8). There are also indications that polyribosomes are associated with a cytoskeletal framework (14,18,19) and that this kind of association may be essential for mRNA translation in the cell (20). This raises the possibility that the p40-containing particles might be involved in the polysomecytoskeleton interaction.…”

Section: Resultsmentioning

confidence: 89%

A 33-kDa polypeptide with homology to the laminin receptor: component of translation machinery.

Auth

Brawerman

1992

Proc. Natl. Acad. Sci. U.S.A.

112

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A 33-kDa polypeptide (termed p40), which shares an antigenic determinant with a laminin receptor and is under translational control, is believed to serve as a precursor to the receptor and to be related to the neoplastic state. The present study of subcellular localization of this protein shows it to be a cytoplasmic component not associated with the plasma membrane. Most of the cellular p40 was found to be associated with polyribosomes as well as with 40S to 60S cytoplasmic particles. Conditions that lead to polysome disruption also caused release of the polysomal form of p40 as smaller particles, and polysome reconstitution was accompanied by uptake of p40 into these structures. Because of the large abundance of this protein in the cells (six to eight copies per ribosome), it is unlikely that it represents a factor that associates with the 40S preinitiation complex. The p40-containing particles appear to represent a newly discovered structure involved in the process of polysome formation.Previous studies in this laboratory have led to the identification of an abundant mRNA species that is under translational control in a variety ofmouse tumor cells (1). The mouse mRNA codes for a polypeptide termed p40 on the basis of its mobility on polyacrylamide gels in the presence of SDS (2, 3). Analysis of a cloned cDNA for the p40 mRNA species showed it to code for a 33-kDa polypeptide (4). A human cDNA clone isolated from a colon carcinoma on the basis of the unusually high abundance of mRNA in the tumor (5) showed nearly complete homology to the mouse p40. A possible relationship between p40 and a laminin-binding protein was suggested by the fact that it contains an octapeptide sequence identical to that of a peptide derived from the 68-to 72-kDa laminin receptor (6). Moreover, a mouse cDNA clone obtained by screening with an antibody to the laminin receptor (7) proved to be essentially identical to the p40 cDNA. A relationship between p40 and the neoplastic state has been proposed (7).The present study deals with characterization of p40 in the cell. We found no evidence for association ofthis protein with membranes in cell extracts. Major portions of p40 are associated with 40S to 60S cytoplasmic particles and with polyribosomes in various cell types. Shifts of p40 between the small particle state and polysomes were observed under conditions that lead to generation or loss of polysomes both in vivo and in cell extracts. We conclude from our results that p40 is part of a cytoplasmic structure that is involved in the translation process.

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“…After allowing the mixture to stand overnight at -20°, the nucleic acids were collected by centrifugation and washed three times with 70% ethanol. The nucleic acids were immediately dissolved with stirring at 30, in 10 ml of a solution containing 10 (14). [3H]uridine for 10 min and the RNA was extracted by the three procedures described under Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Purification of a putative precursor of globin messenger RNA from mouse nucleated erythroid cells.

Kwan¹,

Wood²,

Lingrel³

1977

Proc. Natl. Acad. Sci. U.S.A.

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Nucleated erythroid cells were incubated for 10 min in the presence of [5-Hluridine, and the total RNA was isolated by three different extraction procedures. RNA containing globin messenger RNA sequences was purified from other cellular RNAs by selective hybridization to globin complementary DNA cellulose. Depending upon the extraction procedure employed, 0.4-0.6% ofthe radioactively-labeled total cellular RNA applied to the column annealed to globin comlementary DNA cellulose. (27 Ci/mmol), were added and the incubation continued for an additional 10 min. After the incubation, the cells were chilled on ice, collected by centrifugation, washed once with 10 ml of cold (40) isotonic saline, and resuspended in 2 ml of cold saline. Isolation of RNA Proteinase K Procedure. The cell suspension (0.9 ml) was added dropwise with stirring to 50 ml of a solution (12) containing 30 mM Tris-HCI, (pH 7.4), 100 mM NaCl, 5 mM EDTA, 3% sodium dodecyl sulfate (NaDodSO4), heparin (150 yg/ml) dextran sulfate (100 ,g/ml), 3 mM each of 2' and 3' AMP, CMP, and UMP, and 300 ,ug/ml of proteinase K. The proteinase K was autodigested in the above buffer, without NaDodSO4, for 10 min at 350 prior to use. The cell suspension was incubated for 30 min at 350. After the incubation, 50 ml of the above buffer including NaDodSO4, but without proteinase K were added, followed by 100 ml of buffer-saturated, redistilled phenol and chloroform (1:1, vol/vol). The mixture was stirred at room temperature for 15 min. The aqueous phase was collected by centrifugation at 5000 X g for 5 min at 200.The interphase was re-extracted with extraction buffer minus proteinase K and the combined aqueous phases were re-extracted twice with phenol/chloroform and once with chloroform alone. The aqueous solution was adjusted to 0.3 M NaCl and the nucleic acids were precipitated with 2 volumes of absolute ethanol at -200. After allowing the mixture to stand overnight at -20°, the nucleic acids were collected by centrifugation and washed three times with 70% ethanol. The nucleic acids were immediately dissolved with stirring at 30, in 10 ml of a solution containing 10 mM sodium acetate (pH 5.2), 3 mM MgCI2, heparin (200 ,g/ml), polyvinyl sulfate (30 ,ug/ml), dextran sulfate (200 Ag/ml), 3 mM each of 2' and 3' AMP, CMP, and UMP and 40,g/ml of DNase (Worthington RNase-free) which had been treated with iodoacetate (13). The mixture was stirred at 100 for 20-25 min during which time the viscosity became markedly reduced. The solution was then made 2% in NaDodSO4, 5 mM in EDTA, and 300 ,tg/ml of proteinase K was added to digest the DNase. After a 10 min incubation at 300, sodium acetate (pH 5.2) was added to give a final concentration of 50 mM and an equal volume of phenol/chloroform was then added and the mixture stirred for 5 min at 25°. The aqueous layer was collected, re-extracted first with phenol/chloroform and then twice with chloroform, and the RNA precipitated as described above. The RNA pellet was washed with 70% ethanol, dissolved in water and stored at -700...

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Attachment of Ribosomes to Membranes During Polysome Formation in Mouse Sarcoma 180 Cells

Cited by 63 publications

References 8 publications

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats

The effect of phenobarbitone on protein synthesis by liver polyribosomes in fed and starved rats

A 33-kDa polypeptide with homology to the laminin receptor: component of translation machinery.

Purification of a putative precursor of globin messenger RNA from mouse nucleated erythroid cells.

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