Trans-kingdom conjugation offers a powerful gene targeting: tool in yeast

Gene targeting is one of the powerful techniques used to investigate eukaryotic genes. In a typical eukaryotic microbe, Saccharomyces cerevisiae yeast, we examined trans-kingdom conjugation between Escherichia coli bacterium and yeast as a gene targeting tool. Here, it is shown that trans-kingdom conjugation effectively induced gene replacement even on yeast's target loci (e.g. ura3-52 allele) which is never targeted by conventional transformation. This clearly indicates that trans-kingdom conjugation offers a very powerful gene targeting tool in yeasts. In fact, Southern hybridization analysis of transconjugants distinctly verified the accuracy in the conjugative gene replacement. The efficiency of gene replacement was about 0.4×10⁻⁷ per recipient yeast. This is enough to sustain gene targeting with gene replacement by trans-kingdom conjugation. We also discuss the mechanism of conjugative gene replacement.     

Phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria, and from Escherichia coli: substrate specificity relationship with regard to ATP analogs

Twelve structural analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA synthetases from hen liver cytoplasm and mitochondria, yeast cytoplasm and mitochondria and E. coli. Three compounds are substrates for all five phenylalanyl-tRNA synthetase, three are completely inactive, while the other ATP analogs show differing properties with the different enzymes. Their Km, Ki and V values have been determined. The importance of the amino group in Position 6, the nitrogen in Position 7 and an unsubstituted Position 8 of the purine moiety as well as the supposed anti-conformation of the glycosidic bond and coordination of the magnesium cation to N-7 appear to be conserved through evolution. Bulky substituents on the 2' and 3' of the ribose moiety are generally not tolerated. Graduation of substrate properties of some analogs are similar for the intracellular heterotopic isoenzymes from yeast and hen liver.     

Dynamics of orthophosphate in yeast cytoplasm

Summary Orthophosphate concentration of baker's yeast Saccharomyces cerevisiae was investigated during dynamic conditions. As an example for those dynamics in cell metabolism the transition from glucose limitation to glucose excess (Crabtree-effect) was choosen. As a result of the metabolic switch from complete to partial oxidative metabolism, the cytoplasmic phosphate concentration increased suddenly from 8.4 mM to a maximum of 17.5 mM and transiently decreased to a minimum of 7.0 mM.     

Formation of the yeast mitochondrial membrane. 3. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose depression

The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH₂-cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis.The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide.When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH₂-cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.     

Effect of temperature on protein synthesis and leucine transport by yeast mitochondria

Protein synthesis in yeast mitochondria shows biphasic Arrhenius plots both in vivo and in vitro, with a twofold increase in the activation energy below the transition temperature suggesting a functional association between mitochondrial protein synthesis and the inner membrane. Analysis by gel electrophoresis of mitochondrial translation products labeled in vivo showed that the same proteins are synthesized and then inserted into the membrane above and below the transition temperature of the membrane. The rate of leucine uptake into mitochondria was decreased at least fivefold in the presence of chloramphenicol, suggesting that leucine is used mainly for protein synthesis. In the absence of chloramphenicol, the rate of leucine uptake was always slightly higher but comparable to the incorporation rate of leucine into protein at all temperatures studied, suggesting that the transport of leucine into mitochondria is not rate-limiting for protein synthesis. The ionophore valinomycin or the uncoupler carbonyl phenylhydrazone (CCCP) inhibited 75-80% of the leucine uptake in the presence of chloramphenicol. In addition, the omission of respiratory chain substrates and the ATP-regenerating system led to a 93% inhibition of uptake, suggesting that leucine uptake may occur by an active transport mechanism.     

Protein synthesis in mitochondria from yeast strains carrying nam and mim suppressor genes

Yeast mitochondria isolated from two different wild type strains (gal+ and gal-), whether grown on galactose or glucose, synthesize all mitochondrial polypeptides with similar efficiencies and in proportions approximating those detected in vivo. Mitochondria isolated from mit- mutants synthesize in vitro a mutant pattern of mitochondrial proteins, indistinguishable from the in vivo products. The mutant pattern is restored to the wild type one in mitochondria isolated from pseudorevertant strains carrying an additional nuclear (nam3-1 and R705) or mitochondrial (mim3-1) informational suppressor gene. Suppression is expressed in isolated mitochondria without the obligatory presence of cytosol at the level of both respiratory control and specific polypeptide synthesis. Translation in isolated mitochondria is sensitive to paromomycin. The antibiotic differentiates between translation in mitochondria from wild type strains and that in nam-type gene carrying strains. This strongly suggests that nam-type mutations affect the mitoribosome, enhancing ambiguity of translation, thus allowing for the pseudoreversion of mit- phenotypes.     

t-Loops in yeast mitochondria

Mitochondria of several yeast species contain a linear DNA genome possessing specific terminal DNA structures dubbed mitochondrial telomeres. Several tandemly repeated units and a 5' single-stranded extension characterize mitochondrial telomeres in Candida parapsilosis, Pichia philodendra and Candida salmanticensis. Resemblance of this type of mitochondrial telomeres to typical nuclear telomeres suggests that they might form t-loop structures. Therefore we adopted a protocol for stabilization of potential t-loops in the mtDNA of C. parapsilosis and observed several loops at the ends of the mtDNA. A potential role of t-loops in protection of the ends of mtDNA and/or in mitochondrial telomere dynamics is discussed.     

Effect of tribenzylphosphate on the active phosphate transport and ATP synthesis in yeast mitochondria

Tribenzylphosphate (TBP), a specific inhibitor of the high affinity system for Pi transport in yeast mitochondria, inhibits the active Pi transport measured by the energy-linked swelling. The dependence of the rate of oligomycin sensitive ATP synthesis as a function of the external Pi concentration shows two kinetic systems. The high affinity system, corresponds to the range of the external Pi concentration which stimulates the respiratory rate. TBP inhibits both this system and the state 4 leads to state 3 transition.     

Biogenesis of mitochondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions

The ability in vitro of yeast mitochondrial and microsomal fractions to synthesize lipid de novo was measured. The major phospholipids synthesized from sn-[2-(3)H]glycerol 3-phosphate by the two microsomal fractions were phosphatidylserine, phosphatidylinositol and phosphatidic acid. The mitochondrial fraction, which had a higher specific activity for total glycerolipid synthesis, synthesized phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid, together with smaller amounts of neutral lipids and diphosphatidylglycerol. Phosphatidylcholine synthesis from both S-adenosyl[Me-(14)C]methionine and CDP-[Me-(14)C]choline appeared to be localized in the microsomal fraction.     

A cytochrome c-GFP fusion is not released from mitochondria into the cytoplasm upon expression of Bax in yeast cells

To study Bax-induced release of cytochrome c in vivo, we have expressed a cytochrome c-GFP (green fluorescent protein) fusion in Saccharomyces cerevisiae cells null for the expression of the endogenous cytochrome. We show here that cytochrome c-GFP is efficiently localised to mitochondria and able to function as an electron carrier between complexes III and IV of the respiratory chain. Strikingly, while natural cytochrome c is released into the cytoplasm upon expression of Bax, the cytochrome c-GFP fusion is not. Nevertheless, cells co-expressing Bax and the cytochrome c-GFP fusion die, indicating that mitochondrial release of cytochrome c is not essential for cell death to occur in yeast. The failure to release cytochrome c-GFP is presumed to arise from increased bulk due to the GFP moiety. We propose that in intact yeast cells, Bax-induced release of cytochrome c into the cytoplasm occurs through a selective pore and not as a consequence of the non-specific breakage of the mitochondrial outer membrane.     

Cell-free synthesis of mitochondrial ATPase inhibitor precursor and its transport into yeast mitochondria

ATPase inhibitor protein, which blocks mitochondrial ATPase activity by forming an enzyme-inhibitor complex, was found to be synthesized as a larger precursor in a cell-free translation system directed by yeast mRNA. Other protein factors, which stabilize latent ATPase by binding to the enzyme-inhibitor complex, were also found to be formed as larger precursors. The precursor of ATPase inhibitor protein was transported into isolated yeast mitochondria and was cleaved to the mature peptide in the mitochondria. Impaired mitochondria lacking phosphorylation activity could not convert the precursor to the mature form. Neither antimycin A nor oligomycin alone exhibited a marked effect on the transport-processing of the precursor by intact mitochondria. However, when antimycin A was added with oligomycin, the transport-processing was markedly inhibited. The processing was also strongly inhibited by an uncoupler, carbonylcyanide p-trifluoro-methoxyphenyl hydrazone. The inhibition by the uncoupler was not relieved by ATP added externally. It is concluded that the transport-processing of precursor proteins requires intact mitochondria with a potential difference across the inner membrane.