Mitochondrial translation products during release from glucose repression in Saccharomyces cerevisiae.

Mitochondrial protein synthesis was studied during release from glucose repression in Saccharomyces cerevisiae cells bearing different mitochondrial genomes. The increase in the rate of the synthesis of mitochondrial translation products was analyzed during respiratory induction. Different kinetic patterns were found for strains having a different structure of mitochondrial mosaic genes, even when the nuclear background was the same. A very limited response of the synthesis of the var1 ribosomal protein to inducing conditions was observed.     

Mutations releasing mitochondrial biogenesis from glucose repression in Saccharomyces cerevisiae.

Mutants which exhibit a constitutive glucose-insensitive expression of respiratory activity were selected by use of a triphenyltetrazolium staining technique. These mutants lack carbon catabolite repression, as was demonstrated by measuring cytochromes, the activity of succinate cytochrome c reduction, total cellular respiration, mitochondrial protein, and DNA synthesis. High growth rates of mutant cells in glucose medium and normal fermentative CO2 production exclude the possibility that this carbon catabolite insensitivity of mitochondrial functions is merely due to a decreased utilization of glucose. Accordingly, the activities of the two cytoplasmic enzymes measured, maltase and malate synthase, were glucose repressible to the same extent in the mutants as in the wild type. The mutations are dominant and showed nuclear inheritance. The results are discussed in terms of carbon catabolite-regulated expression of genes involved in the biogenesis of mitochondria.     

High-affinity glucose transport in Saccharomyces cerevisiae is under general glucose repression control.

Saccharomyces cerevisiae mutants defective in growth on low glucose concentration (lgn mutants) were isolated and screened for abnormal glucose transport. Nine complementation groups were identified, falling into two broad groups: those unable to significantly derepress high-affinity (low-Km) glucose uptake (lgn1, lgn4, lgn5, lgn7, and lgn8), and those with elevated repressed levels of high-affinity uptake that either derepress to normal or near normal levels of high-affinity uptake with loss of low-affinity transport (lgn2 and lgn3) or derepress only slightly, appearing to have an intermediate yet constitutive level of high-affinity transport (lgn6 and lgn9). Further analysis of the lgn mutations revealed pleiotropic phenotypes most consistent with the true defect being in regulation or expression of glucose repression and derepression. The kinetics of glucose uptake in strains carrying known mutations preventing derepression of glucose-repressible functions (snf1, snf2, snf4, and snf6) demonstrated that three of these mutations (snf1, snf4, and snf6) were similarly defective in derepression of high-affinity glucose uptake. The snf2 and snf5 mutations had no apparent effect on glucose uptake. Two mutations resulting in constitutive expression of glucose-repressible functions, cid1 and reg1, resulted in constitutive expression of high-affinity glucose uptake. These data support the conclusion that high-affinity glucose uptake in Saccharomyces cerevisiae is under general glucose repression control. The implications of other properties of these mutants are discussed.     

Steady state analysis of mitochondrial RNA after growth of yeast Saccharomyces cerevisiae under catabolite repression and derepression

The steady state levels of mitochondrial rRNAs, 5 tRNAs, the 9 S RNA, and the RNA products from the genes coding for subunits 6 and 9 of the ATP synthase, cytochrome b, and subunit 1 of cytochrome oxidase have been determined after growth of yeast under conditions of respiratory repression or derepression. The analysis indicates that the mitochondrial rRNAs are present in 2000 or 9000 copies/cell in repressed or derepressed yeast, respectively. The levels of the other RNAs also differed to a similar extent, with the exception of the level of the tRNAfMet which differs by only 1.7-fold. The levels of the individual protein coding RNAs varied from 480 copies/cell for the Oli-1 RNA to 100 copies/cell for the Oli-2 RNA under derepressive conditions and from 130 copies/cell to 33 copies/cell for the same RNAs in glucose repressive conditions. The levels of the tRNAs varied even more markedly, ranging from 4200 copies/cell for the tRNAPhe to 240 copies/cell for the tRNACys after growth in derepressive conditions and from 800 copies/cell for the tRNAfMet to 30 copies/cell for the tRNACys of glucose repressed yeast. These results indicate that glucose repression uniformly decreases the levels of the individual mitochondrial RNAs studied. This decrease is related to a lower synthesis of mitochondrial RNA in the glucose repressed cells as compared to derepressed cells.     

mRNA structures influencing translation in the yeast Saccharomyces cerevisiae.

The mRNA sequence and structures that modify and are required for translation of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae were investigated with sets of CYC1 alleles having alterations in the 5' leader region. Measurements of levels of CYC1 mRNA and iso-1-cytochrome c in strains having single copies of altered alleles with nested deletions led to the conclusion that there is no specific sequence adjacent to the AUG initiator codon required for efficient translation. However, the nucleotides preceding the AUG initiator codon at positions -1 and -3 slightly modified the efficiency of translation to an order of preference similar to that found in higher cells. In contrast to large effects observed in higher eucaryotes, the magnitude of this AUG context effect in S. cerevisiae was only two- to threefold. Furthermore, introduction of hairpin structures in the vicinity of the AUG initiator codon inhibited translation, with the degree of inhibition related to the stability and proximity of the hairpin. These results with S. cerevisiae and published findings on other organisms suggest that translation in S. cerevisiae is more sensitive to secondary structures than is translation in higher eucaryotes.     

Association between defects of karyogamy and translation termination in yeast Saccharomyces cerevisiae

Mutants capable of a high frequency of cytoduction (Hfc+) were obtained in a haploid strain of Saccharomyces cerevisiae, suggesting impaired cytogamy. Nine of the 68 Hfc+ mutants showed the antisuppressor effect with respect to mutations of the SUP35 and SUP45 genes, which code for translation termination factors, or to the [PSI+] factor, which is the prion form of Sup35. Cosegregation of the characters "higher frequency of cytoduction" and "antisuppression" was demonstrated for three Hfc+ mutants. One (HFC12-2) of the Hfc+ mutations exerted a dominant antisuppressor effect with respect to [PSI+] and had no effect on [PSI+] maintenance. On the strength of the results, an interaction was assumed for translation termination components and cytoskeleton proteins, which play a role in karyogamy in yeasts.     

Electrophoretic behaviour of yeast mitochondrial translation products.

We have studied the mobility of yeast mitochondrial translation products during electrophoresis on polyacrylamide gels of different composition and found that these polypeptides can be divided into two groups. One, to which subunit II of cytochrome c oxidase belongs, behaves normal as all water-soluble reference proteins. The other, to which cytochrome b and subunits I and III of cytochrome c oxidase belong, shows a free electrophoretic mobility about twice as fast as the first group. Conditions have been found to separate cytochrome c1 from cytochrome b.     

The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae.

Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both promitochondria and mitochondria obtained from O2-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N2) to elaidic (O2)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O2-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.     

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.     

Relationship between cytoplasmic and mitochondrial apparatus of protein synthesis in yeast Saccharomyces cerevisiae

A conditional respiratory deficiency in yeast Saccharomyces cerevisiae is expressed as a result of a nuclear mutation in sup1 and sup2 genes (II and IV chromosomes, respectively), coding for a component of cytoplasmic ribosomes (Ter-Avanesyan et al. 1982). One such strain is studied here in detail. The strain is temperature-dependent and expresses a respiratory deficient phenotype at 20 degrees C but not at 30 degrees C. Moreover, the strain is simultaneously chloramphenicol-dependent and is able to grow on media containing glycerol or ethanol as a sole carbon source only in the presence of the drug. Chloramphenicol has a differential effect on protein synthesis in mitochondria of the parent strain and the mutant. Since chloramphenicol is a ribosome-targeting antibiotic we suggest that the differential effect of the drug on parent and mutant mitochondrial protein synthesis is due to the altered properties of mito-ribosomes of the mutant compared to those of the parent strain. Mitochondria of the mutant synthesize all the mitochondrially encoded polypeptides, however, in significantly lowered amounts. A suggestion is put forward for the existence of a common component (a ribosomal protein) for mito and cyto-ribosomes.     

New genes involved in carbon catabolite repression and derepression in the yeast Saccharomyces cerevisiae.

A mutation causing resistance to carbon catabolite repression in gene HEX2, mutant allele hex2-3, causes an extreme sensitivity to maltose when in combination with the genes necessary for maltose metabolism. This provided a convenient system for the selective isolation of mutations in genes specifically required for maltose metabolism and other genes involved in general carbon catabolite repression. In addition to reversion of the hex2-3 allele, mutations in three other genes were detected. These genes were called CAT1, CAT3, and MUR1 and in a mutated form abolished maltose inhibition caused by mutant allele hex2-3. Mutant alleles cat1 and cat3 also restored normal repression in the presence of the hex2-3 allele. Segregants having only mutant alleles cat1 or cat3 were obtained by tetrad analysis. These segregants could not grow on nonfermentable carbon sources. Mutant alleles of gene CAT1 were allelic to a mutant allele cat1-1 previously isolated (Zimmermann et al., Mol. Gen. Genet. 151:95-103). Such mutants prevented derepression not only of the maltose catabolizing system, the selected property, but also of glyoxylate shunt and gluconeogenic enzymes. However, respiratory activities and invertase formation were not affected under derepressing conditions. cat3 mutants had the same phenotypic properties as cat1 mutants. This showed that carbon metabolism in yeast cells is under a very complex and ramified control of repressing and derepressing genes, which are interdependent.