tRNA binding stabilizes rat liver 60 S ribosomal subunits during treatment with LiCl.

Reboud, Anne-Marie; Dubost, Simone; Buisson, Monique; Reboud, Jean‐Paul

doi:10.1016/s0021-9258(18)43668-x

Cited by 25 publications

(7 citation statements)

References 34 publications

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“…However, in the case of 60S subunits, there are strong arguments which favor the hypothesis that covalent puromycin binding occurs on a specific and functional site: the relatively small distribution of label among the ribosomal proteins and the diminution of the labeling reaction when using antibiotics such as gougerotin and amicetin known to act at the peptidyltransferase center specifically. The results of our competition experiments with tetracycline were recently confirmed by experiments which showed that [3H] tetracycline was directly photoincorporated into those proteins (LIO, L13a, and LI8a) which were found to be at the puromycin-binding sites, either on ribosomes or on isolated 60S subunits, protein L10 being the most labeled one.8 Our recent findings that on isolated 60S subunits protein L10 was protected against salt removal by prior fixation of deacylated tRNA at the P site (Reboud et al, 1980b) and directly photoreacted with deacylated [32P]tRNA when at the P site8 support the tentative conclusion that, among the [3H]puromycin-labeled 60S subunit proteins, at least one, protein L10, forms part of a functional site. This protein was also found by other authors to react, in affinity labeling experiments, with either photoactivable puromycin or aminoacyl-tRNA derivatives (Bóhm et al, 1979;Czernilofsky et al, 1977;Stahl et al, 1979).…”

Section: Discussionsupporting

confidence: 65%

“…The absence of competition with tetracycline is more significant, since this compound does bind to 40S subunits. We recently confirmed that [3H]tetracycline binds to 40S subunit proteins different from those which were found to interact with puromycin.8 Alternative hypotheses can be proposed to explain, at least partly, such a wide distribution of the label among 40S subunit proteins: (1) a high heterogeneity of 40S subunits so that some of them are in different states and therefore are not labeled exactly to the same extent, within the same proteins, and indeed such a heterogeneity of the 40S subunits has been recently observed towards high salt treatment8 while 60S subunits yielded homogeneous species under the same conditions (Reboud et al, 1980b); (2) a proximity of the labeled 40S subunit proteins, as evidenced by Terao et al (1980), for proteins S3-S11, S5-S7-7a, S7-S18, and S23-24, using free subunits and a bifunctional cross-linking reagent;…”

Section: Discussionmentioning

confidence: 99%

“…These sites are located on both subunits, very close to each other. Moreover, protein L10 is easily removable by salt (Reboud et al, 1980b) whereas S3 is not,8 and both of them associate to ribosomal particles at later stages of the maturation process (Auger-Buendia et al, 1979).…”

Section: Discussionmentioning

confidence: 99%

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Photoincorporation of puromycin into rat liver ribosomes and subunits

Am¹,

Dubost²,

Buisson³

et al. 1981

Biochemistry

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[3H]Puromycin was covalently incorporated into rat liver ribosomes and isolated 40S and 60S subunits on irradiation at 254 nm. A study of the concentration dependence of this photolytic incorporation suggested that it arose from specific sites on isolated subunits but also from unspecific ones in the case of ribosomes, these sites being probably located on contaminant nonribosomal proteins. Puromycin was incorporated simultaneously into ribosomal proteins and rRNAs. The results from simultaneous one-dimensional and two-dimensional gel electrophoreses showed a small distribution of label among ribosomal proteins in 60S subunits and in 80S ribosomes, L10 being the most radioactive protein. Some antibiotics, which act on the peptidyltransferase center (amicetin and gougerotin), and also tetracycline competed with this labeling. Therefore, it was concluded that puromycin interaction with protein L10 occurred most likely at a functional site. In the case of free 40S subunits, labeling distribution among proteins was much wider. The possibility that proteins S3 and perhaps S23-24, which were significantly labeled in crude ribosomes too, also belong to a specific site interacting with puromycin is discussed.

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Section: Discussionsupporting

confidence: 65%

Section: Discussionmentioning

confidence: 99%

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Photoincorporation of puromycin into rat liver ribosomes and subunits

Am¹,

Dubost²,

Buisson³

et al. 1981

Biochemistry

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“…GRK2 was purified as previously described (). Preparation of rat liver eEF-2 (95% pure), of 60S and 40S ribosomal subunits by zonal centrifugation, and of recombinant ribosomal proteins P1 and P2 has already been described ( , ). Phosphatidylcholine and phosphatidylinositol 4,5-diphosphate were from Sigma.…”

Section: Methodsmentioning

confidence: 99%

β₂-Adrenergic Receptor Stimulated, G Protein-Coupled Receptor Kinase 2 Mediated, Phosphorylation of Ribosomal Protein P2

et al. 2002

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G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the beta2-adrenergic receptor (beta2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the beta2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the beta2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with K(M) values approximating 1 microM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.

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“…This again rationalizes the presence of two distinct cross-link species. treated with MBS and followed by LiCl, which extracts most ribosomal proteins from the rRNA (Reboud et al, 1980). After extraction, the membranes were floated through a Nycodenz cushion to separate membrane-bound proteins from ribosomal fragments.…”

Section: Rpl17 Is In Proximity To Sec61 In the Ribosome-translocon Complexmentioning

confidence: 99%

A trans-membrane segment inside the ribosome exit tunnel triggers RAMP4 recruitment to the Sec61p translocase

Pool

2009

Journal of Cell Biology

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Membrane protein integration occurs predominantly at the endoplasmic reticulum and is mediated by the translocon, which is formed by the Sec61p complex. The translocon binds to the ribosome at the polypeptide exit site such that integration occurs in a cotranslational manner. Ribosomal protein Rpl17 is positioned such that it contacts both the ribosome exit tunnel and the surface of the ribosome near the exit site, where it is intimately associated with the translocon. The presence of a trans-membrane (TM) segment inside the ribosomal exit tunnel leads to the recruitment of RAMP4 to the translocon at a site adjacent to Rpl17. This suggests a signaling function for Rpl17 such that it can recognize a TM segment inside the ribosome and triggers rearrangements of the translocon, priming it for subsequent TM segment integration.

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tRNA binding stabilizes rat liver 60 S ribosomal subunits during treatment with LiCl.

Cited by 25 publications

References 34 publications

Photoincorporation of puromycin into rat liver ribosomes and subunits

Photoincorporation of puromycin into rat liver ribosomes and subunits

β₂-Adrenergic Receptor Stimulated, G Protein-Coupled Receptor Kinase 2 Mediated, Phosphorylation of Ribosomal Protein P2

A trans-membrane segment inside the ribosome exit tunnel triggers RAMP4 recruitment to the Sec61p translocase

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tRNA binding stabilizes rat liver 60 S ribosomal subunits during treatment with LiCl.

Cited by 25 publications

References 34 publications

Photoincorporation of puromycin into rat liver ribosomes and subunits

Photoincorporation of puromycin into rat liver ribosomes and subunits

β2-Adrenergic Receptor Stimulated, G Protein-Coupled Receptor Kinase 2 Mediated, Phosphorylation of Ribosomal Protein P2

A trans-membrane segment inside the ribosome exit tunnel triggers RAMP4 recruitment to the Sec61p translocase

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β₂-Adrenergic Receptor Stimulated, G Protein-Coupled Receptor Kinase 2 Mediated, Phosphorylation of Ribosomal Protein P2