Stimulation of yeast mitochondrial protein synthesis by postpolysomal supernatants from yeast,rat liver,and Escherichia coli

Isolated yeast mitochondria incubated with a protein-synthesizing mixture containing excess oxidizable substrate, amino acids, MgCl₂, an ATP-regenerating system, and optimal levels of [³H]leucine cease protein synthesis after 30 min. Postpolysomal supernatants from either yeast, rat liver, or Escherichia coli can restore protein synthetic activity to depleted yeast mitochondria; however the addition of bovine serum albumin to the incubation mixture did not restore activity. The restored incorporation activity was sensitive to chloramphenicol, insensitive to cycloheximide, and proportional to the protein concentration of the supernatants. Furthermore, addition of all three high-speed supernatants to isolated mitochondria at time zero stimulated the rate of protein synthesis to a greater extent than when these fractions were added to depleted mitochondria. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the translation products obtained from mitochondria labeled in vitro in the presence of supernatant fractions were identical to the proteins labeled by mitochondria in vivo; however, the synthesis of the bands corresponding to subunit III of cytochrome oxidase, cytochrome b, and VAR-3 was stimulated to the greatest extent. The stimulatory activity in the supernatants was non-dialyzable, insensitive to treatment with ribonuclease A, but completely abolished by pretreatment with trypsin suggesting that the stimulatory factor(s) is of a protein nature. The postpolysomal supernatants did not incorporate amino acids into protein when incubated without mitochondria. These results suggest that the protein synthetic capacity of mitochondria is apparently limited by extramitochondrial proteins which are present in either yeast, rat liver, or E. coli.     

Monocistronic messenger RNA in yeast

We have determined the rate of polypeptide chain synthesis on different size polysomes in yeast. The completion time for the average polypeptide chain in vivo at 23 °C is two minutes by this technique and is in good agreement with values we have determined by other independent methods.These kinetic experiments indicate that the average size of a nascent polypeptide chain on a polysome is directly related to the size of the polysome. This demonstrates that in the simple eucaryotic organism, Saccharomyces cerevisiae, mRNA is monocistronic in the sense that each mRNA molecule codes for one protein molecule which is released intact from the ribosome upon completion. The pattern of amino acid incorporation into Escherichia coli polysomes is distinctly different. These findings have a number of interesting implications for the genetics of the lower eucaryotes and indicate that the cellular mechanisms of control and co-ordination in yeast may differ from those found in procaryotes and may be similar to cellular mechanisms of control for mammalian cells.     

Accessibility of DNA polymerases to repair synthesis during nucleotide excision repair in yeast cell-free extracts

Nucleotide excision repair (NER) removes a variety of DNA lesions. Using a yeast cell-free repair system, we have analyzed the repair synthesis step of NER. NER was proficient in yeast mutant cell-free extracts lacking DNA polymerases (Pol) β, ζ or η. Base excision repair was also proficient without Polβ. Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Polα (pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Polδ (pol3-1) or Pol (pol2-18). Residual repair synthesis was observed in pol3-1 and pol2-18 mutant extracts, suggesting a repair deficiency rather than a complete repair defect. Deficient NER in pol3-1 and pol2-18 mutant extracts was specifically complemented by purified yeast Polδ and Pol, respectively. Deleting the polymerase catalytic domain of Pol (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Pol, but not by purified yeast Polη. These results suggest that efficient repair synthesis of yeast NER requires both Polδ and Pol in vitro, and that the low fidelity Polη is not accessible to repair synthesis during NER.     

Trans-kingdom rescue of Gln-tRNAGln synthesis in yeast cytoplasm and mitochondria

Aminoacylation of transfer RNA^Gln (tRNA^Gln) is performed by distinct mechanisms in different kingdoms and represents the most diverged route of aminoacyl-tRNA synthesis found in nature. In Saccharomyces cerevisiae, cytosolic Gln-tRNA^Gln is generated by direct glutaminylation of tRNA^Gln by glutaminyl-tRNA synthetase (GlnRS), whereas mitochondrial Gln-tRNA^Gln is formed by an indirect pathway involving charging by a non-discriminating glutamyl-tRNA synthetase and the subsequent transamidation by a specific Glu-tRNA^Gln amidotransferase. Previous studies showed that fusion of a yeast non-specific tRNA-binding cofactor, Arc1p, to Escherichia coli GlnRS enables the bacterial enzyme to substitute for its yeast homologue in vivo. We report herein that the same fusion enzyme, upon being imported into mitochondria, substituted the indirect pathway for Gln-tRNA^Gln synthesis as well, despite significant differences in the identity determinants of E. coli and yeast cytosolic and mitochondrial tRNA^Gln isoacceptors. Fusion of Arc1p to the bacterial enzyme significantly enhanced its aminoacylation activity towards yeast tRNA^Gln isoacceptors in vitro. Our study provides a mechanism by which trans-kingdom rescue of distinct pathways of Gln-tRNA^Gln synthesis can be conferred by a single enzyme.     

Defects in triacylglycerol lipolysis affect synthesis of triacylglycerols and steryl esters in the yeast

Tgl3p, Tgl4p and Tgl5p are the major triacylglycerol lipases of the yeast Saccharomyces cerevisiae catalyzing degradation of triacylglycerols stored in lipid droplets. Previous results from our laboratory (Athenstaedt and Daum, 2005, J. Biol. Chem. 280, 37301–37309) demonstrated that a yeast strain lacking all three triacylglycerol lipases accumulates not only triacylglycerols at high amount, but also steryl esters. Here we show a metabolic link between synthesis and mobilization of non-polar lipids. In particular, we demonstrate that a block in tri-acylglycerol degradation in a tgl3?tgl4?tgl5? triple mutant lacking all major triacylglycerol lipases causes marked changes in non-polar lipid synthesis. Under these conditions formation of triacylglycerols is reduced, whereas steryl ester synthesis is enhanced as shown by quantification of non-polar lipids, in vivo labeling of lipids using [¹⁴C]oleic acid and [¹⁴C]acetic acid as precursors, and enzyme analyses in vitro. In summary, this study demonstrates that triacylglycerol metabolism and steryl ester metabolism are linked processes. The importance of balanced storage and degradation of these components for lipid homeostasis in the yeast is highlighted.     

The regulation of RNA synthesis in yeast I: Starvation experiments

Summary The synthesis of tRNA in yeast is shown to be under separate control to that of rRNA during amino acid and nitrogen starvation. Inhibitors of the elongation and termination steps of protein synthesis were found to stimulate the synthesis of tRNA in starved yeast cells. This effect appeared to be due to the trickle-charging of tRNA. Two inhibitors of early steps in the initiation of protein synthesis were found to be unable to stimulate RNA synthesis in starved cells. It is proposed that yeast tRNA synthesis is under autoregulatory control and that the level of tRNA charging and the mRNA-ribosome complex are important components of this control system.     

Cloning of the yeast tyrosine transfer RNA genes in bacteriophage lambda.

Lambda bacteriophage containing yeast tyrosine transfer RNA genes were prepared by molecular recombination. These phage were identified by hybridization of ¹²⁵I-labeled yeast tRNA^Tyr to plaques from lambda-yeast recombinant phage pools. The cloned yeast EcoRI fragments that hybridize to ¹²⁵I-labeled tRNA^Tyr were compared in size with the fragments in total yeast DNA that hybridize to the same probe. These comparisons indicate that seven of the eight different tRNA^Tyr genes have been isolated. Unambiguous evidence that these seven fragments contain tRNA^Tyr coding regions was obtained by showing that they hybridize to aminoacylated [^3H]Tyr-tRNA^Tyr. Only one of the fragments hybridizes to ³²P-labeled total yeast tRNA in the presence of competing unlabeled tRNA^Tyr; the tRNA^Tyr genes, therefore, are not predominantly organized into heteroclusters of tRNA genes.     

Inhibition of RNA synthesis in yeast protoplasts by a peptide factor from Tetrahymena cells

Protoplasts of Schizosaccharomyces pombe, grown on a rich nutrient medium, were treated with a peptide factor isolated from cultures of the protozoan Tetrahymena pyriformis. The peptide factor is known to inhibit RNA synthesis in Tetrahymena. It has now been shown that the peptide factor also inhibits RNA synthesis in yeast protoplasts without affecting protein synthesis.     

Reversible arrest of haploid yeast cells in the initiation of DNA synthesis by a diffusible sex factor

A diffusible substance, α factor, is produced constitutively by haploid yeast cells of α mating type and this factor specifically inhibits the division of a mating type cells. Experiments are presented which demonstrate that α factor arrests a cells as unbudded, mononucleate cells prior to the initiation of DNA synthesis in the cell cycle. Studies with temperature-sensitive mutants defective in one of thirteen different cell cycle functions suggest that although arrested a cells continue to enlarge they do not perform functions required for the next cell cycle. The arrest is reversible and a partially synchronized round of DNA replication is observed upon removal of α factor from arrested cells. We propose that this factor is one element of a regulatory system that functions to assure the synchronization of a and α haploid cell cycles prior to conjugation.     

Ded1p, a conserved DExD/H-box translation factor, can promote yeast L-A virus negative-strand RNA synthesis in vitro

Viruses are intracellular parasites that must use the host machinery to multiply. Identification of the host factors that perform essential functions in viral replication is thus of crucial importance to the understanding of virus–host interactions. Here we describe Ded1p, a highly conserved DExD/H-box translation factor, as a possible host factor recruited by the yeast L-A double-stranded RNA (dsRNA) virus. We found that Ded1p interacts specifically and strongly with Gag, the L-A virus coat protein. Further analysis revealed that Ded1p interacts with the L-A virus in an RNA-independent manner and, as a result, L-A particles can be affinity purified via this interaction. The affinity-purified L-A particles are functional, as they are capable of synthesizing RNA in vitro. Critically, using purified L-A particles, we demonstrated that Ded1p specifically promotes L-A dsRNA replication by accelerating the rate of negative-strand RNA synthesis in vitro. In light of these data, we suggest that Ded1p may be a part of the long sought after activity shown to promote yeast viral dsRNA replication. This and the fact that Ded1p is also required for translating brome mosaic virus RNA2 in yeast thus raise the intriguing possibility that Ded1p is one of the key host factors favored by several evolutionarily related RNA viruses, including the human hepatitis C virus.     

Chemo-enzymatic synthesis of optically pure rivastigmine intermediate using alcohol dehydrogenase from baker's yeast

An efficient and practical synthesis of (S)-rivastigmine intermediate was developed by employing a chemoenzymatic step toward the synthesis of chiral intermediate N-ethyl-N-methyl-carbamic acid-3-(1S-hydroxy-ethyl)-phenyl ester (2) using crude alcohol dehydrogenase from baker's yeast with reduced nucleotide adenosine dinucleotide (NADH) as proton donor has been demonstrated.     

Translesion synthesis of acetylaminofluorene-dG adducts by DNA polymerase zeta is stimulated by yeast Rev1 protein

Translesion synthesis is an important mechanism in response to unrepaired DNA lesions during replication. The DNA polymerase ζ (Polζ) mutagenesis pathway is a major error-prone translesion synthesis mechanism requiring Polζ and Rev1. In addition to its dCMP transferase, a non-catalytic function of Rev1 is suspected in cellular response to certain types of DNA lesions. However, it is not well understood about the non-catalytic function of Rev1 in translesion synthesis. We have analyzed the role of Rev1 in translesion synthesis of an acetylaminofluorene (AAF)-dG DNA adduct. Purified yeast Rev1 was essentially unresponsive to a template AAF-dG DNA adduct, in contrast to its efficient C insertion opposite a template 1,N⁶-ethenoadenine adduct. Purified yeast Polζ was very inefficient in the bypass of the AAF-dG adduct. Combining Rev1 and Polζ, however, led to a synergistic effect on translesion synthesis. Rev1 protein enhanced Polζ-catalyzed nucleotide insertion opposite the AAF-dG adduct and strongly stimulated Polζ-catalyzed extension from opposite the lesion. Rev1 also stimulated the deficient synthesis by Polζ at the very end of undamaged DNA templates. Deleting the C-terminal 205 aa of Rev1 did not affect its dCMP transferase activity, but abolished its stimulatory activity on Polζ-catalyzed extension from opposite the AAF-dG adduct. These results suggest that translesion synthesis of AAF-dG adducts by Polζ is stimulated by Rev1 protein in yeast. Consistent with the in vitro results, both Polζ and Rev1 were found to be equally important for error-prone translesion synthesis across from AAF-dG DNA adducts in yeast cells.     

On the mechanism of premeiotic DNA synthesis in the yeast Saccharomyces cerevisiae

Premeiotic DNA synthesis in synchronously sporulating cultures of the yeast, Saccharomyces cerevisiae, was analysed by sedimentation in alkaline sucrose gradients and by DNA-fibre autoradiography. The gradient profiles of cells pulse-labelled for varying times were essentially identical with those obtained with mitotic cultures, revealing a close resemblance between the meiotic and mitotic replication mechanisms. This was supported by the finding that exposure of meiotic cells to a specific concentration of hydroxyurea led to the accumulation of completed, but unjoined replicons, just as it does in mitotic cells. The results of DNA-fibre autoradiography confirmed that replicons in meiotic cells are the same size (20–180 Kb, averaging around 90 Kb) as in mitotic cells, and assuming replication is bi-directional, replication forks must move at round the same rate as in mitosis, i.e. about 0.7 μm/min.     

Ribosomal RNA from the yeast and mycelial phases ofHistoplasma capsulatum

Purified yeast and mycelial phase rRNA fromHistoplasma capsulatum was heat-formaldehyde deantured and analyzed by polyacrylamide gel electrophoresis. An average difference of 6.9% was seen in electrophoretic migration between the yeast and mycelial phase denatured 17 S rRNAs that was not apparent for the native 17 S rRNAs. This difference was statistically significant and could not be accounted for by random variation among gels electrophoresed in parallel. A significant difference was not found between the 25 S rRNAs of yeast and mycelial phase cells. Molecular weights of 1.28 × 10⁶ for both the yeast and mycelial phase 25 S rRNA, and 0.79 × 10⁶ and 0.73 × 10⁶ for the yeast and mycelial phase 17 S rRNA, were estimated by using denaturing conditions which minimize conformational contributions to electrophoretic migration in polyacrylamide gels. In addition, we find that both the yeast and mycelial phase 17 S and 25 S rRNAs ofH. capsulatum are smaller than their respective counterparts inS. cerevisiae. The results indicate that a structural difference may exist, at least between the 40 S subunits ofH. capsulatum yeast and mycelial phase ribosomes.     

Membrane synthesis, specific lipid requirements, and localized lipid composition changes associated with a positive-strand RNA virus RNA replication protein

Multifunctional RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus, localizes to the cytoplasmic face of endoplasmic reticulum (ER) membranes and induces ER lumenal spherules in which viral RNA synthesis occurs. We previously showed that BMV RNA replication in yeast is severely inhibited prior to negative-strand RNA synthesis by a single-amino-acid substitution in the ole1w allele of yeast Δ9 fatty acid (FA) desaturase, which converts saturated FAs (SFAs) to unsaturated FAs (UFAs). Here we further define the relationships between 1a, membrane lipid composition, and RNA synthesis. We show that 1a expression increases total membrane lipids in wild-type (wt) yeast by 25 to 33%, consistent with recent results indicating that the numerous 1a-induced spherules are enveloped by invaginations of the outer ER membrane. 1a did not alter total membrane lipid composition in wt or ole1w yeast, but the ole1w mutation selectively depleted 18-carbon, monounsaturated (18:1) FA chains and increased 16:0 SFA chains, reducing the UFA-to-SFA ratio from ~2.5 to ~1.5. Thus, ole1w inhibition of RNA replication was correlated with decreased levels of UFA, membrane fluidity, and plasticity. The ole1w mutation did not alter 1a-induced membrane synthesis, 1a localization to the perinuclear ER, or colocalization of BMV 2a polymerase, nor did it block spherule formation. Moreover, BMV RNA replication templates were still recovered from cell lysates in a 1a-induced, 1a- and membrane-associated, and nuclease-resistant but detergent-susceptible state consistent with spherules. However, unlike nearby ER membranes, the membranes surrounding spherules in ole1w cells were not distinctively stained with osmium tetroxide, which interacts specifically with UFA double bonds. Thus, in ole1w cells, spherule-associated membranes were locally depleted in UFAs. This localized UFA depletion helps to explain why BMV RNA replication is more sensitive than cell growth to reduced UFA levels. The results imply that 1a preferentially interacts with one or more types of membrane lipids.     

Regulation of mitochondrial ribosomal RNA synthesis in yeast

Summary Studies were undertaken to determine if mitochondrial rRNA synthesis in yeast is regulated by general cellular stringent control mechanism. Those variables affecting the relaxation of a cycloheximide-induced stringent response as a result of medium-shift-down or tyrosine limitation include: 1) the stage of cell growth, 2) carbon source, 3) strain differences and, 4) integrity of the cell wall. The extent of phenotypic relaxation decreased or was eliminated entirely in a strain dependent manner as cells entered stationary phase of growth or by growth of cells on galactose or in osmotically stabilized spheroplast cultures.Cytoplasmic and mitochondrial RNA species were extracted from regrowing spheroplast cultures subjected to different experimental regimens and analyzed by electrophoresis on 2.5% polyacrylamide gels. Relative rates of synthesis were determined in pulse experiments and normalized by double-label procedures to longterm label material. Tyrosine starvation was found to inhibit synthesis of the large and small rRNA species of both cytoplasmic and mitochondrial rRNAs to about 5–20% of the control values. Chloramphenicol inhibits mitochondrial and cytoplasmic rRNA synthesis to 60–80% of control; however, chloramphenicol addition does not relax the stringent inhibition of either class of rRNAs. Cycloheximide addition results in 70–80% inhibition of synthesis of both cellular species of rRNAs. As noted above, cycloheximide does not relax the stringent response of cytoplasmic rRNA synthesis in spheroplasts, and also does not relax the stringent inhibition of mitochondrial rRNA synthesis. From these studies, we conclude that both cytoplasmic and mitochondrial rRNA synthesis share common control mechanisms related to regulation of protein synthesis by shift-down or amino acid limitation.     

Inhibitory effect of glucose and adenosine 3′,5′-monophosphate on the synthesis of inducible N-acetylglucosamine catabolic enzymes in yeast

Glucose can block the utilization of N-acetylglucosamine in Saccharomyces cerevisiae, a facultative aerobe, but not in Candida albicans, an obligatory aerobe. Furthermore, glucose represses the synthesis of the enzymes of the N-acetylglucosamine catabolic pathway in S. cerevisiae, but not in C. albicans. The results suggest that catabolite repression is present in S. cerevisiae, but not in C. albicans. Cyclic AMP added to S. cerevisiae cells maintained in a glucose medium cannot bring about their release from catabolite repression. On the contrary, the synthesis of inducible enzymes of N-acetylglucosamine pathway was inhibited by cyclic AMP in both the yeasts. This seems to indicate that cyclic AMP can penetrate into the yeast cells. Furthermore, cyclic AMP inhibits protein synthesis, suggesting that protein synthesis in yeast is under cyclic AMP control.     

Temperature-sensitive mutation in yeast mitochondrial ribosome recycling factor (RRF)

The yeast protein Rrf1p encoded by the FIL1 nuclear gene bears significant sequence similarity to Escherichia coli ribosome recycling factor (RRF). Here, we call FIL1 Ribosome Recycling Factor of yeast, RRF1. Its gene product, Rrf1p, was localized in mitochondria. Deletion of RRF1 leads to a respiratory incompetent phenotype and to instability of the mitochondrial genome (conversion to rho^–/rho⁰ cytoplasmic petites). Yeast with intact mitochondria and with deleted genomic RRF1 that harbors a plasmid carrying RRF1 was prepared from spores of heterozygous diploid yeast. Such yeast with a mutated allele of RRF1, rrf1-L209P, grew on a non-fermentable carbon source at 30 but not at 36°C, where mitochondrial but not total protein synthesis was 90% inhibited. We propose that Rrf1p is essential for mitochondrial protein synthesis and acts as a RRF in mitochondria.     

Preferential synthesis of yeast mitochondrial DNA in alpha factor-arrested cells

α factor is a diffusible substance produced by $\text{S. cerevisiae}$ cells of the $\text{α}$ mating type which inhibits cell division (1) and the initiation of nuclear DNA synthesis (2) in cells of the $\text{a}$ mating type. In this report, it is shown that mitochondrial DNA synthesis continues at a normal rate in $\text{a}$ cells for at least 6 hours in the presence of α factor, resulting in a 5-fold increase in the amount of mitochondrial DNA per cell. The continued synthesis of mitochondrial DNA in the absence of nuclear DNA synthesis allows specific labeling of yeast mitochondrial DNA.     

The regulation of RNA synthesis in yeast IV

Summary A study has been made of the regulation of the synthesis of Pl double-stranded (ds) RNA, the genome of the yeast virus-like particle. When yeast protein synthesis is prevented by starvation for a required amino acid or by addition of cycloheximide, the rate of Pl dsRNA synthesis is reduced markedly. During nitrogen starvation the synthesis of Pl dsRNA persists but is accompanied by the degradation of pre-existing molecules. This degradation appears to require the induction of new enzymes and it is likely that the breakdown products are used to enable the cell to complete its division cycle. However, all of the copies of the VLP genome are not degraded in this process, some are conserved and can replenish the amount of Pl dsRNA on return to growth conditions. The controls which must operate on Pl dsRNA synthesis are discussed and compared with those exerted on nuclear RNA synthesis in yeast.Paper III in this series is Elliott and McLaughlin (Molec. Gen. Genet., In press)