Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
Correspondence
Yasuyoshi Sakai
ysakai{at}kais.kyoto-u.ac.jp
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AB104827.
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INTRODUCTION |
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We are studying the physiological role of the glutathione-dependent formaldehyde oxidation pathway using gene-disrupted strains of the methylotrophic yeast Candida boidinii in combination with the methanol-limited chemostat culture technique (Lee et al., 2002; Sakai et al., 1997
). Our previous studies demonstrated that FLD, but not FDH, was essential for growth on methanol, although FDH was necessary for optimal growth on methanol. FGH has been purified to homogeneity from two strains of C. boidinii (Kato et al., 1980
; Neben et al., 1980
). FGH may also play an important role in the regeneration of GSH within cells, since GSH is involved in the detoxification of reactive oxygen species during methanol metabolism (Horiguchi et al., 2001a
; Ubiyvovk et al., 2002
). Distinct from the other two NADH-producing dehydrogenases, the reaction catalysed by FGH, i.e. hydrolysis of S-formylglutathione, might occur non-enzymically or be catalysed by other hydrolytic enzymes in the cells. However, when compared with those of FLD and FDH, our knowledge of the physiological role of FGH in yeast C1 metabolism is very limited, because there have been no reports on gene cloning or mutant analysis of FGH from any methylotrophic yeast strains. We believe this report to be the first to describe (i) the characterization of an FGH-encoding gene from a methylotrophic yeast, (ii) gene-disruption analyses in combination with the methanol-limited chemostat culture technique; and (iii) the subcellular localization of FGH in C. boidinii. The results obtained are discussed from the viewpoint of the physiological roles of FGH and glutathione in yeast C1 metabolism.
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METHODS |
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DNA and RNA methods.
Yeast DNA was isolated by the method of Cryer et al. (1975) or Davis et al. (1980)
. Southern analysis was performed as described previously (Yurimoto et al., 2000a
). Transformation of C. boidinii was performed by the modified lithium acetate method, as described previously (Sakai et al., 1993
). pBluescript II SK+ was from Stratagene. DNA was sequenced with a 7-deaza sequencing kit from Amersham Biosciences and a DNA sequencer model DSQ-2000L from Shimadzu. Total RNA extraction and Northern analysis were performed as described previously (Lee et al., 2002
).
Enzyme assays and protein methods.
We determined FGH activity by measuring the esterase activity towards p-nitrophenyl acetate as a substrate. The activity was determined by monitoring the release of p-nitrophenol photometrically at 420 nm (Degrassi et al., 1999). One enzyme unit was taken as the amount of enzyme releasing 1 µmol p-nitrophenol min-1 at pH 6·0 and 30 °C under the above assay conditions. Protein was determined with a protein assay kit (Bio-Rad) with bovine serum albumin as the standard.
Standard 9 % Laemmli gels (Laemmli, 1970), with the separating gels at pH 9·2, were used. Immunoblotting was performed by the method of Towbin et al. (1979)
using an ECL detection kit (Amersham Biosciences). The anti-GFP antibody was kindly provided by Dr M. Fransen (Katholieke Universiteit, Leuven, Belgium). Subcellular fractionation was performed as described previously (Horiguchi et al., 2001b
). The enzyme activities of AOD and cytochrome c oxidase were assayed as described previously (Sakai et al., 1996
).
Cloning of the C. boidinii FGH1 gene.
Based on the amino acid sequences of highly conserved regions in several FGH-encoding genes (VFYLSGLTC and DHSYYFVS in S. cerevisiae YJL068C) and the preferred codon usage in C. boidinii, two mixed primers, primer 1 and primer 2, were designed (Table 1). Using these two primers and C. boidinii genomic DNA as a template, a 0·7 kb fragment was specifically amplified. Nucleotide sequence analysis showed that the amino acid sequence deduced from the nucleotide sequence of the 0·7 kb fragment was highly similar to those of other FGHs. Therefore, the resultant 0·7 kb fragment was gel-purified and used as a probe for hybridization experiments.
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Construction of an FGH1 gene disruption cassette and one-step gene disruption.
pFGH was digested with NdeI and NcoI to remove an approximately 143 bp fragment of the partial coding sequence of FGH1. The remaining linearized plasmid and the 4·6 kb SacIXhoI fragment of C. boidinii URA3 DNA from pSPR (Sakai & Tani, 1992) were blunt-ended with T4 DNA polymerase and then ligated, yielding FGH1 disruption vector pFGHD. pFGHD had C. boidinii URA3 DNA as a selectable marker and truncated FGH1-flanking sequences. pFGHD was digested with SacI and XhoI, and then used for the transformation of C. boidinii strain TK62. The FGH1-disrupted (fgh1
) strain was caused to revert to uracil auxotrophy after 5-fluoroorotic acid selection, yielding the fgh1
ura3 strain, by our previously described procedure (Sakai & Tani, 1992
). The gene disruption and loss of URA3 were confirmed by genomic Southern analysis using XbaI-digested genomic DNA from each transformant with the 1·0 kb EcoRVNdeI fragment harbouring the 5'-flanking region of FGH1 as the probe (Fig. 2
b).
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RESULTS |
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Only one ORF, of 900 bp, was found. There was no evidence of the presence of an intron in the ORF. From the deduced amino acid sequence, the molecular mass of the protein was calculated to be 33 655 Da (excluding the first Met residue), which corresponded to 31 kDa for the purified enzyme (Kato et al., 1980).
The deduced amino acid sequence of the identified ORF showed 75 %, 62 % and 66 % similarity to those of S. cerevisiae, P. denitrificans and human esterase D, respectively. In the central section, Ser156 of C. boidinii is in the consensus sequence GXSXG around the catalytic serine, typical of all lipases and esterases (Fernandez et al., 2000). Near the C-terminus, conserved His273 is found, which is predicted to be the catalytic histidine residue (Cygler et al., 1993
). The alignment suggested that Asp238 might be the catalytic residue, since this residue is conserved in other FGHs (Derewenda, 1994
). In addition, the extreme C-terminal sequence of FGH from C. boidinii is -SKL, which is a typical peroxisome-targeting signal 1 (PTS1) found in peroxisomal enzymes (Elgersma et al., 1996
; Nishikawa et al., 2000
). Subcellular localization of FGH and the role of C-terminal -SKL are described below. From the results of these and following gene disruption analyses, we concluded that this ORF corresponds to the gene for FGH in C. boidinii, FGH1.
Regulation of FGH1 expression
Previously, we reported that two other genes involved in the glutathione-dependent formaldehyde oxidation pathway, FLD1 and FDH1, were induced by methanol, methylamine and choline. Since the latter two compounds yield formaldehyde when they are used as nitrogen sources, formaldehyde induced the FLD1 and FGH1 expression (Lee et al., 2002; Sakai et al., 1997
). The induction of FLD1 and FDH1 expression was not subject to glucose repression, this feature being distinct from that of the AOD-encoding gene (Yurimoto et al., 2000b
).
The regulation of FGH1 expression by carbon and nitrogen sources was assessed by (i) Northern analysis with FGH1 DNA as a probe or (ii) esterase enzyme assaying with p-nitrophenyl acetate as a substrate (Fig. 3). When the wild-type strain was grown on glucose as the sole carbon source, no FGH1 mRNA was detected (Fig. 3a
). However, a high amount of FGH1 mRNA was detected when cells were grown on methanol. The level of esterase activity in glucose-grown cells was lower than that in methanol-grown cells (Fig. 3b
). Judging from these Northern analyses and the fact that basal levels of esterase activities were detected in the fgh1
strain (Fig. 3b
), the esterase activity of glucose-grown cells was due to some esterase activity other than that of FGH, and the esterase activity induced by methanol could be ascribed to FGH activity. Similarly, induced levels of FGH1 mRNA and esterase activity were observed when cells were grown on methylamine or choline as the sole nitrogen source. Judging from these results, the activity of FGH is induced by both methanol and formaldehyde, and is not subject to glucose repression, as was previously observed for FLD and FDH (Lee et al., 2002
; Sakai et al., 1997
). FGH1 expression was confirmed to be controlled mainly at the mRNA level.
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We compared the growth of the wild-type and fgh1 stains on methanol (as a carbon source), and methylamine and choline (as nitrogen sources) under batch culture conditions. The growth of both strains on glucose or glycerol as the sole carbon source was similar (data not shown). The fgh1
strain could not grow on methanol as the sole carbon source (Fig. 4
a). When methylamine or choline was used as the sole nitrogen source, both the growth rate and growth yield of the fgh1
strain were lower than those of the wild-type strain (Fig. 4b, c
). From these results, FGH was proved to play a significant role in yeast C1 metabolism.
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Subcellular localization of FGH in C. boidinii
So far it has been thought that glutathione-dependent formaldehyde oxidation occurs exclusively in the cytosol, since both FLD and FGH have been shown to be cytosolic. Unexpectedly, FGH from C. boidinii has a C-terminal SKL tripeptide, which is a typical PTS1 sequence. Previously, we showed that a peroxisomal acetylspermidine oxidase also had a C-terminal SKL tripeptide in C. boidinii (Nishikawa et al., 2000). Next, we studied the subcellular localization of FGH in C. boidinii, and examined the physiological significance of the -SKL tripeptide.
Full-length FGH or C-terminal SKL-truncated FGH was tagged with GFP at the N-terminus, integrated into the ura3 locus of the fgh1ura3 strain to form the GFPFGH1 and GFPFGH1
SKL strains, respectively, and then expressed under the constitutive ACT1 promoter. Both strains could complement the growth defect of the fgh1
strain on methanol (Fig. 4a
). Judging from these results, GFP-tagging to FGH did not affect its function, and the C-terminal SKL tripeptide is not essential for its enzymic function nor its physiological function.
Cells of the GFPFGH1 and GFPFGH1SKL strains were grown on methanol medium and then observed by fluorescence microscopy. The GFPAKL strain (Sakai et al., 1998
) showed intrinsic green fluorescent punctate structures and the wild-type strain did not show any fluorescence (Fig. 5
a). The GFPFGH strain exhibited not only cytosolic fluorescence but also punctuate fluorescence, while the GFPFGH1
SKL strain exhibited only cytosolic fluorescence (Fig. 5a
). The subcellular localization of these GFPFGH fusion proteins was further examined by subjecting the GFPFGH1 and GFPFGH1
SKL strains to differential centrifugation, which separated the intracellular components into a cytosolic supernatant (S) and organelle pellet (P), which mainly contained peroxisomes and mitochondria (Fig. 5b
). The activity of AOD, a marker enzyme of peroxisomes, amounted to up to 98 % in the pellet fractions of both strains. The GFP proteins were detected in both the supernatant and pellet fractions in about equal amounts for the GFPFGH1 cells as well as the GFPAKL cells. Furthermore the GFPFGH fusion protein in the pellet fraction was found to be co-localized with AOD on Nycodenz-gradient ultracentrifugation (Fig. 5c
). These results indicted a bimodal distribution of GFPFGH between the cytosol and peroxisomes. In contrast, most GFPFGH
SKL proteins were only detected in the supernatant fraction of the GFPFGH1
SKL cells (Fig. 5b
).
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DISCUSSION |
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Recently, we found that a physiological level of GSH exists within peroxisomes of C. boidinii (Horiguchi et al., 2001a). GSH in peroxisomes is required for the glutathione peroxidase activity of CbPmp20, which detoxifies reactive oxygen species within peroxisomes (Horiguchi et al., 2001a
). On the other hand, formaldehyde non-enzymically reacts with GSH within peroxisomes to form S-hydroxymethylglutathione. Since FLD is not present in peroxisomes, the S-hydroxymethylglutathione formed must be exported to the cytosol. Also, since glutathione reductase activity was not detected in the peroxisome fraction (Horiguchi et al., 2001a
), the oxidized form of glutathione (GS-SG) generated by CbPmp20 within peroxisomes must be exported for regeneration of GSH by glutathione reductase. Therefore, there may be some transport system in peroxisomal membrane for importing and exporting glutathione and its related compounds, at least for GSH, GS-SG and S-hydroxymethylglutathione.
Sequence analysis of FGH1 revealed that FGH has a C-terminal tripeptide SKL, which is the consensus sequence for a peroxisomal-targeting signal (PTS1) (Elgersma et al., 1996; Nishikawa et al., 2000
). By means of subcellular fractionation experiments and observation of GFP-tagged FGHs by fluorescence microscopy, FGH has been shown to exhibit a bimodal distribution between the cytosol and peroxisomes. A similar bimodal distribution was observed for C. boidinii catalase, which was due to the low level of interaction between the C-terminal NKF tripeptide and a PTS1 receptor, Pex5p (Horiguchi et al., 2001b
). In yeast C1 metabolism, the oxidation of methanol to formaldehyde by AOD and subsequent assimilation by dihydroxyacetone synthase occur in peroxisomes. In contrast, the formaldehyde oxidation pathway has been thought to exist exclusively in the cytosol because two other enzymes, FLD and FDH, are localized in the cytosol (Yurimoto et al., 2002
). One possible explanation for the peroxisomal existence of FGH is as follows: a portion of S-formylglutathione, a product of the FLD-catalysed reaction and a substrate for FGH, is transferred from the cytosol to peroxisomes by some transport system mentioned above, and FGH may release GSH within peroxisomes. It might be interesting to determine whether FGHs from other methylotrophic yeast strains are also present in peroxisomes, although Pichia pastoris FGH did not have a putative PTS1 at its C-terminus (unpublished results).
We performed methanol-limited chemostat culture experiments of the knockout strains to study the formaldehyde oxidation pathway enzymes. The accumulation of formaldehyde could be minimized by the use of methanol-limited chemostat conditions with low dilution rates (less than about 0·05 h-1), because the supply of methanol is the rate-limiting factor for methanol metabolism under these conditions. Indeed, the fdh1 strain, which exhibited only weak growth in a batch culture, could grow on methanol under chemostat conditions with a growth yield of one-fourth that of the wild-type strain (0·55 versus 2·3 g dry cell weight g-1) (Sakai et al., 1997
). However, the fld1
strain was unable to grow on methanol even under the same chemostat conditions (Lee et al., 2002
). Our present results showed that the fgh1
strain could grow on methanol as a carbon source and was never washed out from a chemostat culture, although the growth yield of the fld1
strain (0·2 g dry cell weight g-1) was only 10 % that the wild-type strain.
We speculate that these differences in the growth yield among the knockout strains reflect the differences in the contribution of each enzyme to C1 metabolism. The formaldehyde oxidation pathway has two physiological functions, i.e. generation of energy and detoxification of formaldehyde. The difference of growth yield on methanol-limited chemostat conditions among the knockout strains can be explained by the aspect of generation of energy. The wild-type strain could yield 2 mol NADH per 1 mol dissimilated formaldehyde, through the formaldehyde oxidation pathway. While both the fgh1 and fdh1
strains could yield 1 mol NADH per 1 mol dissimilated formaldehyde, the fld1
strain could not yield NADH at all. These results suggest that (1 mol NADH)/(1 mol dissimilated formaldehyde) is sufficient to support growth on methanol and that the NADH molecules generated through the glutathione-dependent formaldehyde oxidation pathway are the main source for energy generation. However, the toxicity of formaldehyde must also be considered. When methylamine or choline was used as the sole nitrogen source, the fgh1
strain showed weaker growth than the wild-type strain (Fig. 4b, c
). In these conditions, the lower growth rate and growth yield in the fgh1
strain were assumed to be caused by the toxicity of formaldehyde, since dissimilation of carbon sources might be the main source of energy. The growth defect of the fgh1
strain on methanol may be also caused by the toxicity of formaldehyde.
While the fgh1 and fdh1
strains were calculated to yield the same amount of NADH, the growth yield of the fgh1
strain was less than half that of the fdh1
strain. In the fgh1
strain, GSH is not released from S-formylglutathione, which would lead to further inefficiency as to formaldehyde oxidation and energy generation. In the wild-type cells as well as those of the fdh1
strain, GSH released from S-formylglutathione would be transported to peroxisomes, where GSH could react with formaldehyde to enter into another cycle of the formaldehyde oxidation pathway or could be used as a reducing agent for CbPmp20 antioxidant activity. In fact, the fgh1
cells had a lower concentration of GSH [0·125 mmol (mg protein-1)] than the fdh1
cells [0·309 mmol (mg protein)-1]. Therefore, besides being a component of the formaldehyde oxidation pathway, FGH seems to play an important role in the regeneration of GSH.
The glutathione-dependent formaldehyde oxidation pathway is found not only in methylotrophs but also in non-methylotrophs (Harms et al., 1996). In general, this pathway is considered to be used for the detoxification of formaldehyde. However, during the evolution of methylotrophs, this pathway might have acquired an energy-generation function as well as that of regulation of the glutathione level in cells.
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ACKNOWLEDGEMENTS |
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Received 26 February 2003;
revised 28 April 2003;
accepted 6 May 2003.
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