The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid.

Gasior, E; Herrera, Flor; Sadnik, Isaac; McLaughlin, Calvin S.; Moldave, Kivie

doi:10.1016/s0021-9258(18)50681-5

Cited by 111 publications

(9 citation statements)

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“…The existence in yeast of cap binding protein(s) and its (their) involvement in translation are not unexpected. It has been shown earlier that yeast mRNAs are capped (Mager et al, 1976;Scripati et al, 1976) and that cap analogues inhibit translation in vitro (Gasior et al, 1979;Tuite et al, 1980;Szczesna & Filipowicz, 1980). Our experiments identify the protein (or one of the proteins) involved.…”

Section: Discussionmentioning

confidence: 57%

“…S. cerevisiae S-100 extracts were prepared from strain GRF-18 according to Gasior et al (1979) and treated with micrococcal nuclease as described (Pelham & Jackson, 1976), except that incubation was at 23 °C for 3-5 min. The composition of protein synthesis incubation mixtures (25 pL) was as described (Gasior et al, 1979) except that the S-100 made up 60% (v/v) of the total reaction mixture and cold methionine was replaced by 5 pCi of [35S]methionine (1000 Ci/mmol). Where indicated, total yeast RNA (see below) was added to a concentration of 3-6 jug/25 pL.…”

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confidence: 99%

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Purification and characterization of protein synthesis initiation factor eIF-4E from the yeast Saccharomyces cerevisiae

et al. 1985

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A 24 000-dalton protein [yeast eukaryotic initiation factor 4E (eIF-4E)] was purified from yeast Saccharomyces cerevisiae postribosomal supernatant by m7GDP-agarose affinity chromatography. The protein behaves very similarly to mammalian protein synthesis initiation factor eIF-4E with respect to binding to and elution from m7GDP-agarose columns and cross-linking to oxidized reovirus mRNA cap structures. Yeast eIF-4E is required for translation as shown by the strong and specific inhibition of cell-free translation in a yeast extract by a monoclonal antibody directed against yeast eIF-4E.

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Section: Discussionmentioning

confidence: 57%

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confidence: 99%

Purification and characterization of protein synthesis initiation factor eIF-4E from the yeast Saccharomyces cerevisiae

et al. 1985

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“…Yeast translation lysates were prepared by a modification of the method described by Gasior et al (1979). 4-6 liters of the S. cere~iae protease deficient strain JB811 was grown in YEP medium containing 2% glucose, to an OI)60o of 2, harvested, washed once with H20, and converted to spbemplasts by digestion with lyticase (10 units/OD) for 30 minutes at 30°C.…”

Section: In Vitro Reactionsmentioning

confidence: 99%

Recycling of proteins from the Golgi compartment to the ER in yeast.

Dean

Pelham

1990

The Journal of Cell Biology

184

108

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Abstract. In the yeast Saccharomyces cerevisiae, the carboxyl terminal sequence His-Asp-Glu-Leu (HDEL) has been shown to function as an ER retention sequence (Pelham, H. R. B., K. G. Hardwick, and M. J. Lewis. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1757-1762). To examine the mechanism of retention of soluble ER proteins in yeast, we have analyzed the expression of a preproalpha factor fusion protein, tagged at the carboxyl terminus with the HDEL sequence. We demonstrate that this fusion protein, expressed in vivo, accumulates intracellularly as a precursor containing both ER and Golgi-specific oligosaccharide modifications. The Golgi-specific carbohydrate modification, which occurs in a SEC18-dependent manner, consists of t~l-6 mannose linkages, with no detectable otl-3 mannose additions, indicating that the transit of the HDEL-tagged fusion protein is confined to an early Golgi compartment. Results obtained from the fractionation of subcellular organelles from yeast expressing HDEL-tagged fusion proteins suggest that the Golgi-modified species are present in the ER. Overexpression of HDEL-tagged preproalpha factor results in the secretion of an endogenous HDEL-containing protein, demonstrating that the HDEL recognition system can be saturated. These results support the model in which the retention of these proteins in the ER is dependent on their receptor-mediated recycling from the Golgi complex back to the ER.T HE complex organization of eukaryotic cells requires mechanisms that direct proteins from their site of synthesis to their site of function. Most secreted and membrane bound proteins are initially directed to the secretory pathway by their cotranslational translocation into the ER. While most of these proteins are subsequently routed to the Golgi body and eventually compartmentalized or secreted, certain proteins remain and function in the ER. Many of the soluble ER proteins in mammalian cells bear a carboxy terminal tetrapeptide sequence, Lys-Asp-Glu-Leu (KDEL), which has been demonstrated to be both necessary and sufficient for the retention of at least one of these proteins (BiP) in the ER (Munro and Pelham, 1987). The growing list of luminal ER proteins that bear this sequence (for review, see Pelham, 1989) implicate it as the distinctive feature that signals retention in the ER.While it is clear that the KDEL sequence functions as an ER retention signal, the mode by which the cellular secretory apparatus recognizes it is less clearly understood. The current model favored for the accumulation of luminal ER proteins envisages a recycling mechanism, in which these proteins are retrieved from a post-ER compartment. The best evidence for this model comes from experiments in animal cells, where it was shown that cathepsin D-KDEL fusion proteins are accessible to post-ER enzymes, and yet accumulate in the ER (Pelham, 1988).Analyses of many aspects of the secretory pathway in the yeast, Saccharomyces cerevisiae, indicate a high degree of conservation with higher eukaryotes. Rather than the sequence ...

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“…Ribosome display (RD), is a completely in vitro method which links the proteins of interest with their mRNA through a stalled ribosome-mRNA-protein complex. RD is performed using cell-free translational systems from bacteria (Hanes and Plückthun 1997), wheat germ (Roberts and Paterson 1973), yeasts (Gasior et al 1979;Tuite et al 1980) or rabbit reticulocytes (Pelham and Jackson 1976). RD is heavily dependent on the integrity of the ribosome-mRNA-protein complexes.…”

Section: Display Technologiesmentioning

confidence: 99%

Technologies for the identification and validation of protein-protein interactions

Pichlerova¹,

Hanes²

2021

gpb

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Proteins are large molecules that play essential roles in all living organisms. In most molecular processes in each cell, proteins usually do not function alone but through physiological interactions with various ligands. The most common interacting molecules for proteins are other proteins, and they act together by protein-protein interactions (PPIs) to create larger protein complexes. The impairment of physiological PPIs or establishing PPIs with pathological proteins often leads to the development of diseases. To bring insights on the knowledge about the physiological functions of proteins in biological processes, and to understand the development and pathogenesis of diseases, numerous qualitative and quantitative methods have been developed. In this review, we summarize the most commonly used methods for studying PPIs, and discuss their advantages and drawbacks.

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The preparation and characterization of a cell-free system from Saccharomyces cerevisiae that translates natural messenger ribonucleic acid.

Cited by 111 publications

References 24 publications

Purification and characterization of protein synthesis initiation factor eIF-4E from the yeast Saccharomyces cerevisiae

Purification and characterization of protein synthesis initiation factor eIF-4E from the yeast Saccharomyces cerevisiae

Recycling of proteins from the Golgi compartment to the ER in yeast.

Technologies for the identification and validation of protein-protein interactions

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