scholarly journals Initiation of Protein Synthesis in Saccharomyces cerevisiae Mitochondria without Formylation of the Initiator tRNA

2000 ◽  
Vol 182 (10) ◽  
pp. 2886-2892 ◽  
Author(s):  
Yan Li ◽  
William B. Holmes ◽  
Dean R. Appling ◽  
Uttam L. RajBhandary
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Carbon Sources ◽  
Normal Growth ◽  
Direct Analysis ◽  
Deletion Strain ◽  
Reading Frame ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Genes Encoding

ABSTRACT Protein synthesis in eukaryotic organelles such as mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl tRNA (fMet-tRNAfMet) for initiation. Here we show that initiation of protein synthesis in yeast mitochondria can occur without formylation of the initiator methionyl-tRNA (Met-tRNAfMet). The formylation reaction is catalyzed by methionyl-tRNA formyltransferase (MTF) located in mitochondria and usesN 10-formyltetrahydrofolate (10-formyl-THF) as the formyl donor. We have studied yeast mutants carrying chromosomal disruptions of the genes encoding the mitochondrial C1-tetrahydrofolate (C1-THF) synthase (MIS1), necessary for synthesis of 10-formyl-THF, and the methionyl-tRNA formyltransferase (open reading frame YBL013W; designated FMT1). A direct analysis of mitochondrial tRNAs using gel electrophoresis systems that can separate fMet-tRNAfMet, Met-tRNAfMet, and tRNAfMet shows that there is no formylation in vivo of the mitochondrial initiator Met-tRNA in these strains. In contrast, the initiator Met-tRNA is formylated in the respective “wild-type” parental strains. In spite of the absence of fMet-tRNAfMet, the mutant strains exhibited normal mitochondrial protein synthesis and function, as evidenced by normal growth on nonfermentable carbon sources in rich media and normal frequencies of generation ofpetite colonies. The only growth phenotype observed was a longer lag time during growth on nonfermentable carbon sources in minimal media for the mis1 deletion strain but not for thefmt1 deletion strain.

Organization of the regulatory region of the yeast CYC7 gene: multiple factors are involved in regulation

1987 ◽  
Vol 7 (9) ◽  
pp. 3252-3259
Author(s):  
T Prezant ◽  
K Pfeifer ◽  
L Guarente
Keyword(s):  
Cytochrome C ◽  
Regulatory Region ◽  
Carbon Sources ◽  
Binding Studies ◽  
Multiple Factors ◽  
Vitro Binding ◽  
Genes Encoding ◽  
Nonfermentable Carbon Source

Regulation of the CYC7 gene of Saccharomyces cerevisiae, encoding iso-2-cytochrome c, was studied. Expression was induced about 20-fold by heme and derepressed 4- to 8-fold by a shift from glucose medium to one containing a nonfermentable carbon source. Deletion analysis showed that induction by heme depends upon sequences between -250 and -228 (from the coding sequence) and upon the HAP1 activator gene, previously shown to be required for CYC1 expression (L. Guarente et al., Cell 36:503-511, 1984). Thus, HAP1 coordinates expression of CYC7 and CYC1, the two genes encoding isologs of cytochrome c in S. cerevisiae. HAP1-18, a mutant allele of HAP1, which increased CYC7 expression more than 10-fold, also acted through sequences between -250 and -228. In vitro binding studies showed that the HAP1 product binds to these sequences (see also K. Pfeifer, T. Prezant, and L. Guarente, Cell 49:19-28, 1987) and an additional factor binds to distal sequences that lie between -201 and -165. This latter site augmented CYC7 expression in vivo. Derepression of CYC7 expression in a medium containing nonfermentable carbon sources depended upon sequences between -354 and -295. The interplay of these multiple sites and the factors that bind to them are discussed.

scholarly journals THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

1972 ◽  
Vol 54 (1) ◽  
pp. 56-74 ◽  
Author(s):  
Paul M. Lizardi ◽  
David J. L. Luck
Keyword(s):  
Protein Synthesis ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Mitochondrial Protein ◽  
Selective Inhibitor ◽  
Mitochondrial Protein Synthesis ◽  
Ribosomal Subunits

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to 14C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-3H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes.

scholarly journals Differences in the products of mitochondrial protein synthesis in vivo in human and mouse cells and their potential use as markers for the mitochondrial genome in human–mouse cell hybrids

1974 ◽  
Vol 144 (1) ◽  
pp. 161-164 ◽  
Author(s):  
Alec Jeffreys ◽  
Ian Craig
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Detailed Comparison ◽  
Cell Types ◽  
Mouse Cell ◽  
Human Cells ◽  
Mitochondrial Protein Synthesis ◽  
Cell Hybrids ◽  
Different Cell Types

The proteins synthesized in the mitochondria of mouse and human cells grown in tissue culture were examined by electrophoresis in polyacrylamide gels. The proteins were labelled by incubating the cells in the presence of [35S]methionine and an inhibitor of cytoplasmic protein synthesis (emetine or cycloheximide). A detailed comparison between the labelled products of mouse and human mitochondrial protein synthesis was made possible by developing radioautograms after exposure to slab-electrophoresis gels. Patterns obtained for different cell types of the same species were extremely similar, whereas reproducible differences were observed on comparison of the profiles obtained for mouse and human cells. Four human–mouse somatic cell hybrids were examined, and in each one only components corresponding to mouse mitochondrially synthesized proteins were detected.

Glutamyl-tRNAGln amidotransferase is essential for mammalian mitochondrial translation in vivo

2014 ◽  
Vol 460 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Lucía Echevarría ◽  
Paula Clemente ◽  
Rosana Hernández-Sierra ◽  
María Esther Gallardo ◽  
Miguel A. Fernández-Moreno ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Synthesis ◽  
Indirect Pathway ◽  
Mitochondrial Translation

We have demonstrated that in mitochondria of mammalian cells the aminoacylation of tRNAGln is produced by an indirect pathway involving the enzyme glutamyl-tRNAGln amidotransferase. Misaminoacylated Glu-tRNAGln is rejected from the ribosomes maintaining the fidelity of the mitochondrial protein synthesis.

scholarly journals Synthesis of mitochondrial protein in the flight muscles of the Colorado beetle. Differentiation in vivo between extra- and intra-mitochondrial contributions to the amino acid incorporation into mitochondrial protein

1973 ◽  
Vol 136 (3) ◽  
pp. 795-802 ◽  
Author(s):  
A. K. M. Bartelink ◽  
C. A. D. De Kort
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Mitochondrial Protein ◽  
Leucine Incorporation ◽  
Amino Acid Incorporation ◽  
Linear Rate ◽  
Synthesis Conditions ◽  
Acid Incorporation ◽  
Flight Muscles

By using cycloheximide, an inhibitor of cytoplasmic protein synthesis, conditions were investigated to estimate in vivo the extra- and intra-mitochondrial contributions to the synthesis of organelle protein in the flight muscles of Colorado beetles. With 4-day-old beetles about 15% of the [14C]leucine incorporation into mitochondrial protein is resistant to the action of cycloheximide. The incorporation into cytosol protein is inhibited by more than 99.5% with cycloheximide. During the first hour after precursor administration the incorporation into mitochondrial protein proceeds, in both the presence and the absence of cycloheximide, at a more-or-less linear rate with time. The cycloheximide-resistant amino acid incorporation is sensitive to the inhibitor of mitochondrial protein synthesis, chloramphenicol. The uncertainties inherent in the use of cycloheximide were discussed in arriving at the conclusion that about 15% of the mitochondrial protein is formed inside the organelle.

THYROXINE STIMULATION OF AMINO ACID INCORPORATION INTO MITOCHONDRIAL PROTEIN: DIFFERENCES BETWEEN IN VIVO AND IN VITRO EFFECTS

1973 ◽  
Vol 72 (4) ◽  
pp. 684-696 ◽  
Author(s):  
Amirav Gordon ◽  
Martin I. Surks ◽  
Jack H. Oppenheimer
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Mitochondrial Protein ◽  
Leucine Incorporation ◽  
Mitochondrial Protein Synthesis ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Stimulation Of

ABSTRACT The in vivo and in vitro stimulation of rat hepatic mitochondrial protein synthesis by thyroxine (T4) was compared. In confirmation of Buchanan & Tapley (1966). T4 added to isolated mitochondria rapidly stimulated [14C] leucine incorporation into mitochondrial protein. The in vitro stimulation was reversed after T4 was removed by incubating the mitochondria with bovine serum albumin (BSA). The decrease in T4 stimulation of protein synthesis appeared proportional to the T4 removed by BSA. Thus, it appears probable that exchangeable T4 controls the in vitro system. In contrast, the increase in mitochondrial protein synthesis which was observed 3 to 4 days after pretreatment of hypothyroid rats with labelled and non-radioactive T4 was not reversed by BSA treatment. Moreover, mitochondrial radioactivity could not be extracted with albumin. The in vivo phenomenon does not, therefore, appear to be related to exchangeable hormone in the mitochondria. Furthermore, the estimated quantity of T4 associated with mitochondria after in vivo stimulation was at least two orders of magnitude less than that required to produce comparable stimulation of mitochondrial protein synthesis in vitro. These findings strongly suggest that in vitro and in vivo stimulation of amino acid incorporation by T4 may be mediated by different biochemical mechanisms.

scholarly journals The Mere Lack of rT Modification in Initiator tRNA Does Not Facilitate Formylation-Independent Initiation inEscherichia coli

2001 ◽  
Vol 183 (24) ◽  
pp. 7397-7402 ◽  
Author(s):  
Swapna Thanedar ◽  
T. K. Dineshkumar ◽  
Umesh Varshney
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Normal Growth ◽  
Assay System ◽  
Inescherichia Coli ◽  
Initiator Trna

ABSTRACT Formylation of initiator methionyl-tRNA is essential for normal growth of eubacteria. However, under special conditions, it has been possible to initiate protein synthesis with unformylated initiator tRNA even in eubacteria. Earlier studies suggested that the lack of ribothymidine (rT) modification in initiator tRNA may facilitate initiation in the absence of formylation. In this report we show, by using trmA strains of Escherichia coli(defective for rT modification) and a sensitive in vivo initiation assay system, that the lack of rT modification in the initiators is not sufficient to effect formylation-independent initiation of protein synthesis.

scholarly journals The lability of the products of mitochondrial protein synthesis in Saccharomyces cerevisiae. A novel method for protein half-life determination

1978 ◽  
Vol 170 (3) ◽  
pp. 569-576 ◽  
Author(s):  
G Y Bakalkin ◽  
S L Kalnov ◽  
A V Galkin ◽  
A S Zubatov ◽  
V N Luzikov
Keyword(s):  
Protein Synthesis ◽  
Half Life ◽  
Mitochondrial Protein ◽  
Double Labelling ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Novel Method ◽  
The Difference ◽  
First Time

A method for the determination of the half-life of mitochondrial translation products in yeast in vivo is proposed. The method uses inhibitors of cytoplasmic and mitochondrial protein synthesis and is based on double-labelling pulse-chase techniques, the second label being used to estimate ‘post-incorporation’ during the ‘chase’. For the first time the difference between post-incroporation and the widely known recycling of the label is considered. These studies show that, in the turnover of mitochondrial translation products, the problem is of post-incorporation into mitochondria (especially from the cell sap) is predominant. The results obtained with this procedure indicate that the half-life of the products of mitochondrial protein synthesis in yeast at the late-exponential phase is about 60 min. The results suggest that mitochondrial transplantation products are subject to proteolysis to acid-soluble forms.

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