Expression level of methanol-inducible peroxisomal proteins and peroxisome morphology are affected by oxygen conditions and mitochondrial respiratory pathway function in the methylotrophic yeast Candida boidinii.

In the methylotrophic yeast, Candida boidinii, methanol-inducible peroxisomal proteins, for example alcohol oxidase (AOD), dihydroxyacetone synthase (DAS), and peroxisomal glutathione peroxidase (Pmp20), were induced only under aerobic conditions, while expression of PMP47 encoding peroxisomal integral membrane protein Pmp47 was independent of oxygen conditions. Expression of the methanol-inducible peroxisomal enzymes was repressed by inhibition of the mitochondrial respiratory chain. In the respiratory-deficient (ρ0) mutant strain, their induction was at very low levels despite the presence of oxygen, whereas the expression of PMP47 was unaffected. Taken together, these facts indicate that C. boidinii can sense oxygen conditions, and that mitochondrial respiratory function may have a profound effect on induction of methanol-inducible gene expression of peroxisomal proteins. Peroxisome morphology was also affected by oxygen conditions and respiratory function. Under hypoxic conditions or respiration-inhibited conditions, cells induced by methanol contained small peroxisomes, indicating that peroxisome biogenesis and the protein import machinery were not affected by oxygen conditions but that peroxisome morphology was dependent on induction of peroxisomal matrix proteins.

Novel physiological roles for glutathione in sequestering acetaldehyde to confer acetaldehyde tolerance in Saccharomyces cerevisiae.

In this work, we identified novel physiological functions of glutathione in acetaldehyde tolerance in Saccharomyces cerevisiae. Strains deleted in the genes encoding the enzymes involved in glutathione synthesis and reduction, GSH1, GSH2 and GLR1, exhibited severe growth defects compared to wild-type under acetaldehyde stress, although strains deleted in the genes encoding glutathione peroxidases or glutathione transferases did not show any growth defects. On the other hand, intracellular levels of reduced glutathione decreased in the presence of acetaldehyde in response to acetaldehyde concentration. Moreover, we show that glutathione can trap a maximum of four acetaldehyde molecules within its molecule in a non-enzymatic manner. Taken together, these findings suggest that glutathione has an important role in acetaldehyde tolerance, as a direct scavenger of acetaldehyde in the cell.

Alteration of ethanol tolerance caused by the deficiency in the genes associated with histone deacetylase complex in budding yeast.

Upon exposure to 8% ethanol, survival and growth of yeast strains deficient in histone deacetylase complex genes was examined. Of the 18 mutants tested, the Δsir3 and Δsir4 strains showed higher resistance to ethanol, while the Δrco1, Δhos3, Δhda2, and Δhst1 strains were more sensitive than the wild type. Furthermore, these ethanol-resistant patterns varied under aerobic and anaerobic culture conditions.

Molecular characterization of the PEX14 gene from the methylotrophic yeast Pichia methanolica.

In this study, we describe the molecular characterization of the PmPEX14 gene encoding the peroxisomal membrane protein from the methylotrophic yeast Pichia methanolica. The pex14Δ strain of P. methanolica lost its ability to grow on methanol and oleate but grew normally on glucose. Disruption of the PmPEX14 caused a decrease in the activities of peroxisomal methanol-metabolizing enzymes and mislocalization of those proteins into the cytosol and vacuole. Taken together, these findings show that PmPex14p has an essential physiological role in methanol metabolism in P. methanolica.

Transcription factor Stb5p is essential for acetaldehyde tolerance in Saccharomyces cerevisiae.

Transcription factor Stb5p, previously known as one of the multidrug resistance gene regulators in Saccharomyces cerevisiae, was shown here to play an essential role in acetaldehyde tolerance. A mutant strain, Δstb5 exhibited increased acetaldehyde sensitivity, and failed to induce genes such as GND1, TKL1 and TAL1 involved in the pentose phosphate pathway (PPP) upon acetaldehyde stress. Using this strain it was revealed that Stb5p acts as a repressor for PGI1 encoding glucose-6-phosphate isomerase under acetaldehyde stress. In reverse, over-expression of Stb5p reinforced acetaldehyde tolerance to the yeast. Furthermore, various deletion mutants of the genes involved in glycolysis showed increased acetaldehyde tolerance compared to the wild-type strain. From these results, it was suggested that Stb5p participates in acetaldehyde tolerance by regulating expression of the PPP genes and PGI1, and that down-regulation of glycolytic pathway may lead to vitalization of PPP and to increased acetaldehyde tolerance.

The peroxisomal catalase gene in the methylotrophic yeast Pichia methanolica.

In this paper, we describe the CTA1 gene, which encodes a peroxisomal catalase in the methylotrophic yeast Pichia methanolica. The P. methanolica CTA1 gene (PmCTA1) comprises a 1,530-bp open reading frame corresponding to a protein of 510 amino acid residues, and its deduced amino acid sequence shows high similarity to those of Cta1ps from other methylotrophic yeasts (about 79%). Expression of PmCTA1 in a peroxisomal catalase-depleted (Cbcta1Delta) Candida boidinii strain restored the methylotrophic growth of the host strain, while the expression of PmCTA1-DeltaSRL, which lacks peroxisome targeting signal type 1, did not. In P. methanolica, expression of PmCTA1 was induced when cells were grown on peroxisome-inducing carbon sources, viz., methanol, oleate, and D-alanine. Taken together, these results indicate that PmCTA1 encodes a functional peroxisomal catalase in P. methanolica.

Molecular characterization of two genes with high similarity to the dihydroxyacetone synthase gene in the methylotrophic yeast Pichia methanolica.

The methylotrophic yeast Pichia methanolica possesses two genes, PmDAS1 and PmDLP1, whose amino acid sequences show high similarity to dihydroxyacetone synthase (DAS), the formaldehyde-fixing enzyme for methanol metabolism within the peroxisome. The PmDAS1 and PmDLP1 genes encode 709 and 707 amino acid residues respectively, and PmDas1p contains a type-1 peroxisomal targeting signal (PTS1), while PmDlp1p does not. Upon phylogenetic analysis, PmDas1p fit into the DAS group with other DASs, while PmDlp1p was grouped with the DAS-like proteins (DLP) of non-methylotrophic yeasts and fungi, a branch of the phylogenetic tree independent of the DAS and transketolase (TK) groups. While expression of PmDAS1 restored the methylotrophic growth of the Candida boidinii das1Delta strain, the PmDLP1 and PmDAS1-DeltaPTS1 genes did not. Taken together, these results indicate that PmDAS1 encodes a functional DAS and has an indispensable role in methanol metabolism, and that PmDlp1p share a common, as yet uncharacterized function in P. methanolica as well as in non-methylotrophic yeasts and fungi.

Acetaldehyde tolerance in Saccharomyces cerevisiae involves the pentose phosphate pathway and oleic acid biosynthesis.

To identify genes responsible for acetaldehyde tolerance, genome-wide screening was performed using a collection of haploid Saccharomyces cerevisiae strains deleted in single genes. The screen identified 49 genes whose deletion conferred acetaldehyde sensitivity, and these were termed the genes required for acetaldehyde tolerance. We focused on six of these genes required for acetaldehyde tolerance, ZWF1, GND1, RPE1, TKL1 and TAL1, which encode enzymes in the pentose phosphate pathway (PPP), and OAR1, which encodes for NADPH-dependent 3-oxoacyl-(acyl-carrier-protein) reductase. These genes were not only responsible for acetaldehyde tolerance but also turned out to be induced by acetaldehyde. Moreover, the content of oleic acid was remarkably increased in yeast cells under acetaldehyde stress, and supplementation of oleic acid into the media partially alleviated acetaldehyde stress-induced growth inhibition of strains disrupted in the genes required for acetaldehyde tolerance and OLE1. Taken together, our data suggest that the supply of NADPH and the process of fatty acid biosynthesis are the key factors in acetaldehyde tolerance in the yeast, and that oleic acid plays an important role in acetaldehyde tolerance.

Molecular characterization of the PEX5 gene encoding peroxisomal targeting signal 1 receptor from the methylotrophic yeast Pichia methanolica.

In this study, we describe the molecular characterization of the PEX5 gene encoding the peroxisomal targeting signal 1 (PTS1) receptor from the methylotrophic yeast Pichia methanolica. The P. methanolica PEX5 (PmPEX5) gene contains a open reading frame corresponding to a gene product of 646 amino acid residues, and its deduced amino acid sequence shows a high similarity to those of Pex5ps from other methylotrophic yeasts. Like other Pex5ps, the PmPex5p possesses seven repeats of the TPR motif in the C-terminal region and three WXXXF/Y motifs. A strain with the disrupted PEX5 gene (pex5Delta) lost its ability to grow on peroxisome-inducible carbon sources, methanol and oleate, but grew normally on glucose and glycerol. Disruption of PmPEX5 caused a drastic decrease in peroxisomal enzyme activities and mislocalization of GFP-PTS1 and some peroxisomal methanol-metabolizing enzymes in the cytosol. Expression of the PmPEX5 gene was regulated by carbon sources, and it was strongly expressed by peroxisome-inducible carbon sources, especially methanol. Taken together, these findings show that PmPex5p has an essential physiological role in peroxisomal metabolism of P. methanolica, including methanol metabolism, and in peroxisomal localization and activation of methanol-metabolizing enzymes, e.g. AOD isozymes, DHAS and CTA.

Peroxisomal metabolism is regulated by an oxygen-recognition system through organelle crosstalk between the mitochondria and peroxisomes.

In the present study using Pichia methanolica, it was found that expressions of methanol-metabolic enzymes were strictly regulated by the presence of oxygen, and that induction of alcohol oxidase (AOD) isozymes was completely dependent on oxygen concentrations. A proportion of AOD-isozyme species responded to oxygen conditions, e.g. in a low oxygen condition, Mod1p was dominant, but with an increase in the oxygen concentration, the ratio of Mod2p increased. The K(m) value of Mod1p for oxygen was ca. one-seventh lower than that of Mod2p (0.47 and 3.51 mM, respectively). This shows that Mod1p is suitable at low oxygen concentrations and Mod2p at high oxygen concentrations. Also, zymogram changes for AOD isozymes were observed by inhibition of respiratory chain activity. These indicated that P. methanolica has the ability to recognize oxygen conditions and the respiratory chain should participate in the sensor for available oxygen. These facts indicate that there is organelle crosstalk between mitochondria and peroxisomes through nucleus gene regulation in order to control the consumption balance of available oxygen between the mitochondrial respiratory chain and peroxisomal AODs.

Distribution, diversity and regulation of alcohol oxidase isozymes, and phylogenetic relationships of methylotrophic yeasts.

In this study, we attempted to classify the methylotrophic yeasts based on diversities of alcohol oxidase (AOD), i.e. zymogram patterns and partial amino acid sequences. According to zymogram patterns for AOD, members of the methylotrophic yeasts separate into two major lineages, one group involving strains having a single AOD and the other group, including Pichia methanolica, Candida pignaliae and C. sonorensis, showing nine AOD isozymes. Based on partial amino acid sequences of AOD, the methylotrophic yeasts could be divided into five groups, and this classification agrees mostly with grouping based on 26S domain D1/D2 rDNA nucleotide sequences, except for some strains. Moreover, the strains having AOD isozymes constitute one group with P. trehalophila, P. glucozyma and Pichia sp. strain BZ159, although these strains are divided into two types, based on amino acid sequences of second AODs. On the other hand, these AOD isozymes consist of two subunits; the first subunits are induced not only by methanol but also by glycerol and pectin, although the second subunits are mainly induced by methanol. These data indicate that AOD isozymes and second AOD genes distribute widely in several methylotrophic yeasts in the natural environment, and second AOD genes may have evolved as methylotrophic genes that can adapt to the environmental conditions of higher methanol concentrations.

Molecular characterization of the Tim9 homologue from the methylotrophic yeast Pichia methanolica.

In this paper we describe molecular characterization of the TIM9 gene encoding the essential mitochondrial inner-membrane protein in the methylotrophic yeast Pichia methanolica. PmTIM9 contains two exons corresponding to a gene product of 89 amino acid residues and a 140 bp intron. The deduced amino acid sequence exhibited high identity to those of other yeast Tim9ps, and possessed two CX(3)C motifs that contained two cysteine residues conserved among small Tim family proteins. Moreover, PmTIM9 had the ability to partially suppress the temperature sensitivity of Saccharomyces cerevisiae strain tim9-3, suggesting that PmTIM9 is a functional homologue of the ScTIM9 gene.