Immunologically related proteins in cytoplasmic and mitochondrial ribosomes of yeast Saccharomyces cerevisiae

Summary Ribosomal proteins from the cytoplasm and mitochondria of the yeast Saccharomyces cerevisiae were compared by immunoblotting techniques. Antibodies raised against cytoplasmic ribosomal proteins cross-react with five mitochondrial ribosomal proteins, four of which are located in the large and one in the small mitochondrial subunits. The possible existence of common ribosomal proteins for cytoplasmic and mitochondrial ribosomes is discussed.Abbreviations cyto cytoplasmic - mito mitochondrial     

Overlapping roles of the cytoplasmic and mitochondrial redox regulatory systems in the yeast Saccharomyces cerevisiae

Thioredoxins are small, highly conserved oxidoreductases which are required to maintain the redox homeostasis of the cell. Saccharomyces cerevisiae contains a cytoplasmic thioredoxin system (TRX1, TRX2, and TRR1) as well as a complete mitochondrial thioredoxin system, comprising a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). In the present study we have analyzed the functional overlap between the two systems. By constructing mutant strains with deletions of both the mitochondrial and cytoplasmic systems (trr1 trr2 and trx1 trx2 trx3), we show that cells can survive in the absence of both systems. Analysis of the redox state of the cytoplasmic thioredoxins reveals that they are maintained independently of the mitochondrial system. Similarly, analysis of the redox state of Trx3 reveals that it is maintained in the reduced form in wild-type cells and in mutants lacking components of the cytoplasmic thioredoxin system (trx1 trx2 or trr1). Surprisingly, the redox state of Trx3 is also unaffected by the loss of the mitochondrial thioredoxin reductase (trr2) and is largely maintained in the reduced form unless cells are exposed to an oxidative stress. Since glutathione reductase (Glr1) has been shown to colocalize to the cytoplasm and mitochondria, we examined whether loss of GLR1 influences the redox state of Trx3. During normal growth conditions, deletion of TRR2 and GLR1 was found to result in partial oxidation of Trx3, indicating that both Trr2 and Glr1 are required to maintain the redox state of Trx3. The oxidation of Trx3 in this double mutant is even more pronounced during oxidative stress or respiratory growth conditions. Taken together, these data indicate that Glr1 and Trr2 have an overlapping function in the mitochondria.     

Mitochondrial mutants of the yeast Saccharomyces cerevisiae showing resistance in vitro to chloramphenicol inhibition of mitochondrial protein synthesis

Two cytoplasmic genetic mutants of yeast, genetically separable by recombination, displaying high levels of chloramphenicol resistance have been isolated. Protein synthesis in isolated mitochondria of mutant [$\text{cap 2-r}$] is almost completely resistant to chloramphenicol inhibition while that in mitochondria of mutant [$\text{cap 1-r}$] is partially resistant. Biochemical differences between the two mutants were confirmed by studies of chloramphenicol inhibition of aerobic adaptation of anaerobically grown cells. The mutants appear to contain altered mitochondrial ribosomes.     

Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in Saccharomyces cerevisiae

Similar to other eukaryotes, yeasts have parallel pathways of one-carbon metabolism in the cytoplasm and mitochondria and have folylpolyglutamate synthetase activity in both compartments. The gene encoding folylpolyglutamate synthetase is MET7 (also referred to as MET23) on chromosome XV and appears to encode both the cytoplasmic and mitochondrial forms of the enzyme. In order to determine the metabolic roles of both forms of folylpolyglutamate synthetase, we disrupted the met7 gene and determined that the strain is a methionine auxotroph and an adenine and thymidine auxotroph when grown in the presence of sulfanilamide. The met7 mutant becomes petite under normal growth conditions but can be maintained with a grande phenotype if the strain is tup and all media are supplemented with dTMP. A met7 gly1 strain is auxotrophic for glycine when grown on glucose but prototrophic when grown on glycerol. A met7 ser1 strain cannot use glycine to suppress the serine auxotrophy of the ser1 phenotype. A met7 shm2 strain is nonviable. In order to disrupt just the mitochondrial folylpolyglutamate synthetase activity, we constructed mutants with an inactivated chromosomal MET7 gene complemented by genes that express only cytoplasmic folylpolyglutamate synthetase, including the Lactobacillus casei folC gene and the yeast MET7 gene with its mitochondrial leader sequence deleted (MET7Deltam). All the genes providing cytoplasmic folylpolyglutamate synthetase complemented the methionine auxotrophy as well as the synthetic lethality of the shm2 strain and the synthetic glycine auxotrophy of the gly1 strain. The strains lacking the mitochondrial folylpolyglutamate synthetase had longer doubling times than the isogenic wild-type strains but retained the function of the mitochondrial folate-dependent enzymes to produce formate, serine, and glycine. Mutants complemented by the bacterial folC gene or by the MET7Deltam gene on a 2mu plasmid remained grande without the tup mutation and supplementation and dTMP. Mutants complemented by the MET7Deltam gene integrated in single copy became petites under those conditions, indicating a deficiency in dTMP production but this is likely due to lower expression of cytoplasmic folylpolyglutamate synthetase by the MET7Deltam gene.     

Inhibition of mitochondrial protein synthesis in vivo by erythromycin in Schizosaccharomyces pombe and Saccharomyces cerevisiae

Summary In the presence of erythromycin (0.01 mg/ml) growth of Schizosaccharomyces pombe in non-fermentable substrate (glycerol) is reduced to 5–15% of the control without erythromycin, whereas growth in fermentable substrate (5% glucose) is left unaffected by concentrations up to 5 mg/ml. The reduction of growth under derepressed conditions is paralleled by inhibition of the formation of cytochromes a·a₃ and b. Mitochondrial protein synthesis is inhibited to about 50% in Schizosaccharomyces pombe and to about 90% in Saccharomyces cerevisiae. These results support the hypothesis that inhibition of mitochondrial protein synthesis is the primary effect of erythromycin.     

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.     

Inorganic polyphosphates of different fractions in the mutant yeast Saccharomyces cerevisiae with impaired mitochondrial ATP synthesis

Impaired synthetase function of the mitochondrial ATPase induced by mutation in the ATP22 gene results in decreased accumulation of inorganic polyphosphates in the stationary growth phase of the yeast Saccharomyces cerevisiae grown on glucose. The content of polyphosphates in the mutant strain in this phase is 2.5 times lower than in the parent strain. This difference is most pronounced for the acid-soluble polyP1 fraction and the alkali-soluble polyP3 fraction. Polyphosphate chain length in mutant cells is less than in the parent cells in both the acid-soluble polyP1 and in the salt-soluble polyP2 fractions. The mutation had no effect on polyphosphates content in the mitochondria.     

Cloning and characterization of the mitochondrial phosphate transport protein gene from the yeast Saccharomyces cerevisiae.

We have cloned the gene of the Saccharomyces cerevisiae phosphate transport protein (PTP), a member of the mitochondrial anion transport protein gene family. As PTP has a blocked N-terminus, we prepared three peptides. Oligonucleotides, based on their sequences, were used to screen a Yep24-housed genomic library. A total of 2073 bases of clone Y22 code for a 311 amino acid protein (Mr 32,814), which has similarities to the anion transport proteins: a triplicate gene structure and 6 hydrophobic segments. Typical for PTP, the triplicate gene structure possesses the X-Pro-X-(Asp/Glu)-X-X-(Lys/Arg)-X-(Arg/Lys)-X (X is an unspecified amino acid) motif and the very high homology only between the first and second repeat. The 6 hydrophobic segments harbor most of the 116 amino acids that are conserved between the yeast and the beef proteins. An N-terminal-extended signal sequence, as found in the beef protein, is absent. The yeast protein has about 33% fewer basic and acidic amino acids and five fewer Cys residues than the beef protein. The protein is insensitive to N-ethylmaleimide since Cys-42 (beef) has been replaced with a Thr. Mersalyl sensitivity has been retained and must be due to one of its three cysteines. Among these three cysteines, only Cys-28, located in the first hydrophobic segment, is conserved between the yeast and the beef protein.     

Proteolysis of products of mitochondrial protein synthesis in isolated mitochondria of Saccharomyces cerevisiae yeasts]

Products of mitochondrial protein synthesis were specifically labeled with 3H-leucine in the presence of cycloheximide at the end of the exponential phase of yeast aerobic growth on glucose. The mitochondria isolated from these cells lost 37-40% of the label from the protein fraction during 60 min incubation at 35 degrees, which was accompanied by the accumulation of 3H-leucine in TCA-soluble fraction. This process was suppressed by phenyl-methyl sulfonyl fluoride and p-chloromercuriphenyl sulfonate, the inhibitors of proteases, and could thus be considered as the proteolysis of the products of mitochondrial protein synthesis. The proteolysis was ATP dependent and was stimulated by puromycine which is known to induce the removal of incomplete polypeptides from mitochondrial ribosomes. A body of indirect evidence allows a suggestion to be made that the observed proteolysis can hardly be due to the action of cytoplasmic proteinases.     

Metabolic effects of mislocalized mitochondrial and peroxisomal citrate synthases in yeast Saccharomyces cerevisiae

Genes CIT1 and CIT2 from Saccharomyces cerevisiae encode mitochondrial and peroxisomal citrate synthases involved in the Krebs tricarboxylic acid (TCA) cycle and glyoxylate pathway, respectively. A Deltacit1 mutant does not grow on acetate, despite the presence of Cit2p that could, in principle, bypass the resulting block in the TCA cycle. To elucidate this absence of cross-complementation, we have examined the ability of Cit1p to function in the cytosol, and that of Cit2p to function in mitochondria. A cytosolically localized form of Cit1p was also incompetent for restoration of growth of a Deltacit1 strain on acetate, suggesting that mitochondrial localization of Cit1p is essential for its function in the TCA cycle. Cit2p was able, when mislocalized in mitochondria, to restore a wild-type phenotype in a strain lacking Cit1p. We have purified these two isoenzymes as well as mitochondrial malate dehydrogenase, Mdh1p, and have shown that Cit2p was also able to mimic Cit1p in its in vitro interaction with Mdh1p. Models of Cit1p and Cit2p structures generated on the basis of that of pig citrate synthase indicate very high structural and electrostatic surface potential similarities between the two yeast isozymes. Altogether, these data indicate that metabolic functions may require structural as well as catalytic roles for the enzymes.     

Purification and characteristics of a mitochondrial endonuclease from the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae contains a membrane-bound mitochondrial nuclease. The enzyme was purified nearly 500-fold from sphaeroplasts of the organism by differential centrifugation, differential solubilization, heparin-agarose chromatography, and gel filtration. A final specific activity of 98 mumol min-1 (mg of protein)-1 was obtained. The enzyme required further purification to achieve homogeneity. Two peaks of activity were obtained after gel filtration with apparent molecular weights of 140000 and 57000. Otherwise, these two components have nearly identical characteristics. Without detergent the enzyme is insoluble and has very low activity. Zwittergent 3-14 or Triton X-100 in the presence of KCl could be used to solubilize and activate the enzyme. A number of other detergents were much less effective in solubilizing or activating the nuclease. The enzyme requires Mg2+ for activity, and this can be replaced to some degree by Mn2+ but not by Ca2+ or Zn2+. It is most active at pH 6.5-7.0 and degrades the substrate to small oligonucleotides with 5'-phosphate ends. The relative rates of hydrolysis were 100 for poly(A), 31 for ssDNA, 19 for RNA, 2.1 for dsDNA, and less than or equal to 0.2 for poly(C). Under the assay conditions used the enzyme appears to constitute about 90% of the total nuclease activity of the cell. The enzyme is unstable, especially at neutral and alkaline pH.     

Comparative electrophoretic study on proteins from mitochondrial and cytoplasmic ribosomes of Saccharomyces cerevisiae

By sodium dodecyl sulfate-acrylamide gel electrophoresis, proteinsof yeast mitochondrial ribosome were shown to be different fromthose of the cytoplasmic counterpart. Fourteen bands found withmitochondrial ribosome did not show any correspondence withthose of the cytoplasmic type. Further, relative amounts ofproteins with their molecular weight more than 50,000 were muchgreater in mitochondrial ribosome, while proteins with theirmolecular weight less than 27,000 were abundant in cytoplasmictype. (Received January 16, 1975; )     

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.     

Characterization of an estrogen-binding protein in the yeast Saccharomyces cerevisiae

This paper further characterizes the estrogen-binding protein we have described in the cytosol of the yeast Saccharomyces cerevisiae. [3H]Estradiol was used as the radioprobe, and specific binding of cytosol fractions was measured by chromatography on Sephadex minicolumns. Other 3H-steroids did not exhibit specific binding. [3H]Estradiol binding was destroyed by treatment with trypsin, but not RNase, DNase, or phospholipase; N-ethylmaleimide substantially decreased the binding. The yeast did not metabolize estradiol added to the medium, and extraction and chromatography of the bound moiety showed it to be unmetabolized estradiol. Scatchard analysis of cytosol from both a and alpha mating types as well as the a/alpha diploid cell revealed similar binding properties: an apparent dissociation constant or Kd(25 degrees) for [3H]estradiol of 1.6-1.8 nM and a maximal binding capacity or Nmax of approximately 2000-2800 fmol/mg of cytosol protein. Gel exclusion chromatography on Sephacryl S-200 and high performance liquid chromatography suggested a Stokes radius of approximately 30 A. Sucrose gradient centrifugation showed a sedimentation coefficient of approximately 5 S, and the complex did not exhibit ionic dependent aggregation. The estrogen binder in S. cerevisiae differed in its steroidal specificities from classical mammalian estrogen receptors in rat uterus. 17 beta-Estradiol was the best competitor, 17 alpha-estradiol had about 5% the activity, and diethylstilbestrol exhibited negligible binding affinity as did tamoxifen, nafoxidine, and the zearalenones. In summary, a high affinity, stereospecific, steroid-selective binding protein has been demonstrated in the cytosol of the simple yeast S. cerevisiae. We speculate that this molecule may represent a primitive hormone receptor system, possibly for an estrogen-like message molecule.