Preparation of a mRNA‐Dependent Cell‐Free Translation System from Whole Cells of <i>Saccharomyces cerevisiae</i>

Hofbauer, Roland; Fessl, Friederike; Hamilton, Barbara; Ruis, Helmut

doi:10.1111/j.1432-1033.1982.tb05867.x

Cited by 46 publications

(18 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The experimental set up utilized for the studies described below consisted of a particle-free (100000 x g, S100) or a particulate (microsomal) translation system (30000 x g, S30), isolated from a yeast lysate according to [13], and a messenger RNA encoding yeast prepro-a-factor (pp-a-F). Prepro-afactor mRNA was generated by in vitro transcription of the MFal gene (see Materials and Methods).…”

Section: A Particulate Lysate From Yeast Translates Prepro-a-factor Mmentioning

confidence: 99%

“…Cell-free translation extracts were prepared according to Hofbauer et al [13]. Briefly, a suspension of yeast cells (5 g wet weight in 12.5ml lysis buffer) was shaken in an SS34 centrifuge tube with 50 g acid-washed glass beads (0.45 -0.5 mm in diameter, Braun-Melsungen) for 3 min with intermittent cooling on ice.…”

Section: Preparation Of Yeast Cell-free Translation Extractsmentioning

confidence: 99%

See 1 more Smart Citation

Subcellular location of enzymes involved in the N‐glycosylation and processing of asparagine‐linked oligosaccharides in Saccharomyces cerevisiae

Tillmann

Günther²,

Schweden³

et al. 1987

European Journal of Biochemistry

View full text Add to dashboard Cite

A particulate translation system isolated from the yeast Succhuromyces cerevisiae was shown to translate faithfully in-vitro-transcribed mRNA coding for a mating hormone precursor (prepro-a-factor mRNA) and to N-glycosylate the primary translation product after its translocation into the lumen of the microsomal vesicles. Glycosylation of its three potential sugar attachment sites was found to be competitively inhibited by acceptor peptides containing the consensus sequence Asn-Xaa-Thr, supporting the view that the glycan chains are N-glycosidically attached to the prepro-a-factor polypeptide. The accumulation in the presence of acceptor peptides of a membrane-specific, unglycosylated translation product (pp-a-Fo) differing in molecular mass from a cytosolically located, protease-K-sensitive a-factor polypeptide (Pp-tx-FcyJ by about 1.3 kDa, suggests that, in contrast to previous reports, a signal sequence is cleaved from the mating hormone precursor on/after translocation. This conclusion is supported by the observation that the multiply glycosylated a-factor precursor is cleaved by endoglucosaminidase H to a product with a molecular mass smaller than the primary translation product ppa-Fcyt but larger than the membrane-specific pp-a-Fo.Translation and glycosylation experiments carried out in the presence of various glycosidase inhibitors ( e g 1-deoxynojirimycin, N-methyl-1-deoxynojirimyin and 1-deoxymannojirimycin) indicate that the N-linked oligosaccharide chains of the glycosylated prepro-a-factor species are extensively processed under the in vitro conditions of translation. From the specificity of the glycosidase inhibitors applied and the differences in the molecular mass of the glycosylated translation products generated in their presence, we conclude that the glycosylation-competent microsomes contain trimming enzymes, most likely glucosidase I, glucosidase I1 and a trimming mannosidase, which process the prepro-a-factor glycans down to the (Man)8(GlcNAc)z stage. Furthermore, several arguments strongly suggest that these three enzymes, which apparently represent the full array of trimming activities in yeast, are exclusively located in the lumen of microsomal vesicles derived from endoplasmic reticulum membranes. N-Glycosylation of proteins is assumed to be a cotranslational event which occurs shortly after or during the translocation of the nascent polypeptide chain from its cytosolic site of synthesis into the luminal space of the endoplasmic reticulum. This reaction is catalysed by membrane-bound N-glycosyltransferases which transfer the (Glc)3(Man)9(GlcNAc)z oligosaccharide from dolichyl diphosphate onto specific asparagine residues within the polypeptide chain. A necessary, though in itself not sufficient,

show abstract

Section: A Particulate Lysate From Yeast Translates Prepro-a-factor Mmentioning

confidence: 99%

Section: Preparation Of Yeast Cell-free Translation Extractsmentioning

confidence: 99%

Subcellular location of enzymes involved in the N‐glycosylation and processing of asparagine‐linked oligosaccharides in Saccharomyces cerevisiae

Tillmann

Günther²,

Schweden³

et al. 1987

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Catalase T mRNA was selected from total yeast RNA isolated as described previously (26) (27) or S. cerevisiae (16). Characterization of translation products by immunoadsorption with catalase antibodies.…”

mentioning

confidence: 99%

Isolation of the catalase T structural gene of Saccharomyces cerevisiae by functional complementation.

Spevak¹,

Fessl²,

Rytka³

et al. 1983

Mol. Cell. Biol.

View full text Add to dashboard Cite

The catalase T structural gene of Saccharomyces cerevisiae was cloned by functional complementation of a mutation causing specific lack of the enzyme (cttl). Catalase T-deficient mutants were obtained by UV mutagenesis of an S. cerevisiae strain bearing the cas1 mutation, which causes insensitivity of catalase T to glucose repression. Since the second catalase protein of S. cerevisiae, catalase A, is completely repressed on 10% glucose, catalase T-deficient mutant colonies could be detected under such conditions. A cttl mutant was transformed with an S. cerevisiae gene library in plasmid YEp13. Among the catalase T-positive clones, four contained overlapping DNA fragments according to restriction analysis. Hybridization selection of yeast mRNA binding specifically to one of the cloned DNAs, translation of this mRNA in cell-free protein synthesis systems, and demonstration of catalase T protein formation by specific immunoadsorption showed that the catalase T structural gene had been cloned. By subcloning, the gene was located within a 3.5-kilobase S. cerevisiae DNA fragment. As in wild-type cells, catalase T synthesis in cttl mutant cells transformed with plasmids containing this fragment is sensitive to glucose repression. By DNA-RNA hybridization, catalase T transcripts were shown to be present in oxygen-adapting cells but absent from heme-deficient cells.

show abstract

“…To test the functional importance of these molecular interactions in transcript-specific translation initiation, we carried out in vitro translation assays using luciferase reporter genes (Hofbauer et al 1982;Hussain and Leibowitz 1986;Wang et al 2013). We generated a monocistronic reporter construct by cloning the 5 ′ UTR of Pab1 mRNA in-frame with the firefly luciferase open reading frame (ORF) under control of the T7 promoter.…”

Section: Sbp1 Inhibits Both Cap-dependent and Cap-independent Translamentioning

confidence: 99%

Sbp1 modulates the translation of Pab1 mRNA in a poly(A)- and RGG-dependent manner

Brandariz-Núñez

Zeng

Lam

et al. 2017

RNA

View full text Add to dashboard Cite

RNA-binding protein Sbp1 facilitates the decapping pathway in mRNA metabolism and inhibits global mRNA translation by an unclear mechanism. Here we report molecular interactions responsible for Sbp1-mediated translation inhibition of mRNA encoding the polyadenosine-binding protein (Pab1), an essential translation factor that stimulates mRNA translation and inhibits mRNA decapping in eukaryotic cells. We demonstrate that the two distal RRMs of Sbp1 bind to the poly(A) sequence in the 5 ′ ′ ′ ′ ′ UTR of the Pab1 mRNA specifically and cooperatively while the central RGG domain of the protein interacts directly with Pab1. Furthermore, methylation of arginines in the RGG domain abolishes the protein-protein interaction and the inhibitory effect of Sbp1 on translation initiation of Pab1 mRNA. Based on these results, the underlying mechanism for Sbp1-specific translational regulation is proposed. The functional differences of Sbp1 and RGG repeats alone on transcript-specific translation were observed, and a comparison of the results suggests the importance of remodeling the 5 ′ ′ ′ ′ ′ UTR by RNA-binding proteins in mRNA translation.

show abstract

Preparation of a mRNA‐Dependent Cell‐Free Translation System from Whole Cells of Saccharomyces cerevisiae

Cited by 46 publications

References 16 publications

Subcellular location of enzymes involved in the N‐glycosylation and processing of asparagine‐linked oligosaccharides in Saccharomyces cerevisiae

Subcellular location of enzymes involved in the N‐glycosylation and processing of asparagine‐linked oligosaccharides in Saccharomyces cerevisiae

Isolation of the catalase T structural gene of Saccharomyces cerevisiae by functional complementation.

Sbp1 modulates the translation of Pab1 mRNA in a poly(A)- and RGG-dependent manner

Contact Info

Product

Resources

About