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We investigated the role of FAD2, which was predicted to encode a fatty acid desaturase of the n-alkane-assimilating yeast Yarrowia lipolytica. Northern blot analysis suggested that FAD2 transcription was upregulated at low temperature or in the presence of n-alkanes or oleic acid. The FAD2 deletion mutant grew as well as the wild-type strain on glucose, n-alkanes, or oleic acid at 30 °C, but grew at a slower rate at 12 °C, when compared to the wild-type strain. The growth of the FAD2 deletion mutant at 12 °C was restored by the addition of 18:2, but not 18:1, fatty acids. The amount of 18:2 fatty acid in the wild-type strain was increased by the incubation at 12 °C and in the presence of n-octadecane. In contrast, 18:2 fatty acid was not detected in the deletion mutant of FAD2, confirming that FAD2 encodes the Δ12-fatty acid desaturase. These results suggest that Δ12-fatty acid desaturase is involved in the growth of Y. lipolytica at low temperature.

Endocytosis is vital for hyphal tip growth in filamentous fungi and is involved in the tip localization of various membrane proteins. To investigate the function of a Wiskott–Aldrich syndrome protein (WASP) in endocytosis of filamentous fungi, we identified a WASP ortholog-encoding gene, wspA, in Aspergillus nidulans and characterized it. The wspA product, WspA, localized to the tips of germ tubes during germination and actin rings in the subapical regions of mature hyphae. wspA is essential for the growth and functioned in the polarity establishment and maintenance during germination of conidia. We also investigated its function in endocytosis and revealed that endocytosis of SynA, a synaptobrevin ortholog that is known to be endocytosed at the subapical regions of hyphal tips in A. nidulans, did not occur when wspA expression was repressed. These results suggest that WspA plays roles in endocytosis at hyphal tips and polarity establishment during germination.

Yarrowia lipolytica possesses twelve ALK genes, which encode cytochromes P450 in the CYP52 family. In this study, using a Y. lipolytica strain from which all twelve ALK genes had been deleted, strains individually expressing each of the ALK genes were constructed and their roles and substrate specificities were determined by observing their growth on n-alkanes and analyzing fatty acid metabolism. The results suggested that the twelve Alk proteins can be categorized into four groups based on their substrate specificity: Alk1p, Alk2p, Alk9p, and Alk10p, which have significant activities to hydroxylate n-alkanes; Alk4p, Alk5p, and Alk7p, which have significant activities to hydroxylate the ω-terminal end of dodecanoic acid; Alk3p and Alk6p, which have significant activities to hydroxylate both n-alkanes and dodecanoic acid; and Alk8p, Alk11p, and Alk12p, which showed faint or no activities to oxidize these substrates. The involvement of Alk proteins in the oxidation of fatty alcohols and fatty aldehydes was also analyzed by measuring viability of the mutant deleted for twelve ALK genes in medium containing dodecanol and by observing growth on dodecanal of a mutant strain, in which twelve ALK genes were deleted along with four fatty aldehyde dehydrogenase genes. It was suggested that ALK gene(s) is/are involved in the detoxification of dodecanol and the assimilation of dodecanal. These results imply that genes encoding CYP52-family P450s have undergone multiplication and diversification in Y. lipolytica for assimilation of various hydrophobic compounds.

Saccharomyces cerevisiae strain Hm-1 is a yeast isolated from the flower of cotton rosemallow. This yeast is used for the production of Seishu, a traditional Japanese refined sake., Saccharomyces cerevisiae strain Hm-1 is a yeast isolated from the flower of cotton rosemallow. This yeast is used for the production of Seishu, a traditional Japanese refined sake. Here, we report the strain’s draft genome sequence. With this genomic information, the brewing characteristics of the strain can be better understood.

To elucidate the mechanism of acyl chain remodeling at the sn-1 position of phosphatidylcholine (PC), we investigated acyl chain introduction using a newly synthesized 1-hydroxy-2-hexadecyl-sn-glycero-3-phosphocholine (HHPC) in Saccharomyces cerevisiae. HHPC is incorporated into yeast cells and converted to a PC species containing acyl residues of 16 or 18 carbons. The efficiency of palmitoleic acid introduction to HHPCin vitro is lower in the reaction with the extract from the deletion mutant of ALE1, which encodes a membrane-bound O-acyltransferase, than in that with extracts from the wild-type strain. In addition, deletion of ALE1 causes reductions in the molecular species containing acyl residues in HHPC. These results reveal that ALE1 is involved in acyl chain transfer to the sn-1 position of HHPC in yeast.

In the n-alkane-assimilating yeast Yarrowia lipolytica, the transcription of ALK1, encoding cytochrome P450, that catalyses n-alkane hydroxylation is activated by a complex composed of Yas1p and Yas2p via a promoter element, ARE1, in response to n-alkanes. An Opi1-family transcription factor, Yas3p, represses the transcription by binding to Yas2p in the nucleus when cultured in glucose-containing medium, but it is localized to the ER, presumably through interaction with acidic phospholipids, phosphatidic acid and/or phospho inositides, when cultured in n-alkane-containing medium. Here, to elucidate the mechanisms regulating the localization of Yas3p, point and deletion mutants of Yas3p were constructed and analysed. The substitution of Trp(360) and Cys(361) by Arg abrogated the localization of Yas3p to the ER and decreased ARE1-mediated transcriptional activation by n-alkane. A Yas3p truncation mutant consisting of residues 259-422 did not bind to acidic phospholipids, but it was localized to the ER in the presence of n-alkane, implying the acidic-phospholipid-independent recruitment of this mutant to the ER in response to n-alkane. The W360R and C361R substitutions in this truncation mutant abolished its localization to the ER. The results suggest that these residues are implicated in the acidic phospholipid-independent interaction of Yas3p to the ER.

A protein kinase C of Aspergillus nidulans, PkcA, is required for cell wall integrity (CWI) and is considered a major component of the regulating pathway. To investigate whether PkcA regulates the transcription of cell wall-related genes, we constructed strains expressing pkcA(R429A) that encodes an activated form of PkcA. The mRNA levels of most chitin synthase genes and an α-glucan synthase gene, agsB, were increased when pkcA(R429A) expression was induced. These mRNA increases were not observed or were only partially observed, in a deletion mutant of rlmA, an ortholog of RLM1 that encodes a transcription factor in the CWI pathway in Saccharomyces cerevisiae. In addition, in a pkcA temperature-sensitive mutant under heat stress, the mRNA levels of some chitin synthase genes and agsB did not increase. These results suggest that PkcA is involved in CWI maintenance through the transcriptional regulation of cell wall-related genes.

Sterols are present in eukaryotic membranes and significantly affect membrane fluidity, permeability, and microdomain formation. They are synthesized in the endoplasmic reticulum (ER) and transported to other organelles and the plasma membrane. Sterols play important roles in the biogenesis and maintenance of mitochondrial membranes. However, the mechanisms underlying ER-to-mitochondrion sterol transport remain to be identified. Here, using purified yeast membrane fractions enriched in ER and mitochondria, we show that the oxysterol-binding protein homologs encoded by the OSH genes in the yeast Saccharomyces cerevisiae mediate sterol transport from the ER to mitochondria. Combined depletion of all seven Osh proteins impaired sterol transport from the ER to mitochondria in vitro; however, sterol transport was recovered at different levels upon adding one of the Osh proteins. Of note, the sterol content in the mitochondrial fraction was significantly decreased in vivo after Osh4 inactivation in a genetic background in which all the other OSH genes were deleted. We also found that Osh5–Osh7 bind cholesterol in vitro. We propose a model in which Osh proteins share a common function to transport sterols between membranes, with varying contributions by these proteins, depending on the target membranes. In summary, we have developed an in vitro system to examine intracellular sterol transport and provide evidence for involvement of Osh proteins in sterol transport from the ER to mitochondria in yeast.

In the n-alkane assimilating yeast Yarrowia lipolytica, n-alkanes are oxidized to fatty acids via fatty alcohols and fatty aldehydes, after which they are utilized as carbon sources. Here, we show that four genes (HFD1-HFD4) encoding fatty aldehyde dehydrogenases (FALDHs) are involved in the metabolism of n-alkanes in Y. lipolytica. A mutant, in which all of four HFD genes are deleted (Δhfd1-4 strain), could not grow on n-alkanes of 12-18 carbons; however, the expression of one of those HFD genes restored its growth on n-alkanes. Production of Hfd2Ap or Hfd2Bp, translation products of transcript variants generated from HFD2 by the absence or presence of splicing, also supported the growth of the Δhfd1-4 strain on n-alkanes. The FALDH activity in the extract of the wild-type strain was increased when cells were incubated in the presence of n-decane, whereas this elevation in FALDH activity by n-decane was not observed in Δhfd1-4 strain extract. Substantial FALDH activities were detected in the extracts of Escherichia coli cells expressing the HFD genes. Fluorescent microscopic observation suggests that Hfd3p and Hfd2Bp are localized predominantly in the peroxisome, whereas Hfd1p and Hfd2Ap are localized in both the endoplasmic reticulum and the peroxisome. These results suggest that the HFD multigene family is responsible for the oxidation of fatty aldehydes to fatty acids in the metabolism of n-alkanes, and raise the possibility that Hfd proteins have diversified by gene multiplication and RNA splicing to efficiently assimilate or detoxify fatty aldehydes in Y. lipolytica.

CTP:phosphoethanolamine cytidylyltransferase (ECT) is a key enzyme in the CDP-ethanolamine branch of the Kennedy pathway, which is the primary pathway of phosphatidylethanolamine (PE) synthesis in mammalian cells. Here, the enzymatic properties of recombinant human ECT (hECT) were characterized. The catalytic reaction of hECT obeyed Michaelis–Menten kinetics with respect to both CTP and phosphoethanolamine. hECT is composed of two tandem cytidylyltransferase (CT) domains as ECTs of other organisms. The histidines, especially the first histidine, in the CTP-binding motif HxGH in the N-terminal CT domain were critical for its catalytic activity in vitro, while those in the C-terminal CT domain were not. Overexpression of the wild-type hECT and hECT mutants containing amino acid substitutions in the HxGH motif in the C-terminal CT domain suppressed the growth defect of the Saccharomyces cerevisiae mutant of ECT1 encoding ECT in the absence of a PE supply via the decarboxylation of phosphatidylserine, but overexpression of hECT mutants of the N-terminal CT domain did not. These results suggest that the N-terminal CT domain of hECT contributes to its catalytic reaction, but C-terminal CT domain does not.

Interorganelle phospholipid transfer is critical for eukaryotic membrane biogenesis. In the yeast Saccharomyces cerevisiae, phosphatidylserine (PS) synthesized by PS synthase, Pss1, in the endoplasmic reticulum (ER) is decarboxylated to phosphatidylethanolamine (PE) by PS decarboxylase, Psd1, in the ER and mitochondria or by Psd2 in the endosome, Golgi, and/or vacuole, but the mechanism of interorganelle PS transport remains to be elucidated. Here we report that Sfh1, a member of Sec14 family proteins of S. cerevisiae, possesses the ability to enhance PE production by Psd2. Overexpression of SFH1 in the strain defective in Psd1 restored its growth on non-fermentable carbon sources and increased the intracellular and mitochondrial PE levels. Sfh1 was found to bind various phospholipids, including PS, in vivo. Bacterially expressed and purified Sfh1 was suggested to have the ability to transport fluorescently labeled PS between liposomes by fluorescence dequenching assay in vitro. Biochemical subcellular fractionation suggested that a fraction of Sfh1 localizes to the endosome, Golgi, and/or vacuole. We propose a model that Sfh1 promotes PE production by Psd2 by transferring phospholipids between the ER and endosome.

In the n-alkane assimilating yeast Yarrowia lipolytica, the expression of ALK1, encoding a cytochrome P450 that catalyzes terminal mono-oxygenation of n-alkanes, is induced by n-alkanes. The transcription of ALK1 is regulated by a heterocomplex that comprises the basic helix-loop-helix transcription activators, Yas1p and Yas2p, and binds to alkane-responsive element 1 (ARE1) in the ALK1 promoter. An Opi1 family transcription repressor, Yas3p, represses transcription by binding to Yas2p. Yas3p localizes in the nucleus when Y. lipolytica is grown on glucose but localizes to the endoplasmic reticulum (ER) upon the addition of n-alkanes. In this study, we showed that recombinant Yas3p binds to the acidic phospholipids, phosphatidic acid (PA) and phosphoinositides (PIPs), in vitro. The ARE1-mediated transcription was enhanced in vivo in mutants defective in an ortholog of the Saccharomyces cerevisiae gene PAH1, encoding PA phosphatase, and in an ortholog of SAC1, encoding PIP phosphatase in the ER. Truncation mutation analyses for Yas3p revealed two regions that bound to PA and PIPs. These results suggest that the interaction with acidic phospholipids is important for the n-alkane-induced association of Yas3p with the ER membrane.

In aquatic invertebrates, particularly marine gastropods, organotin compounds induce irreversible sexual abnormality in females, which is termed imposex, at very low concentrations. Organotin compounds are agonists for nuclear receptors such as RXRs and PPARgamma. However, the imposex phenomenon has not been reported to act as an antagonist on estrogen receptors in other species, including vertebrates and invertebrates. In order to gain insights into the antagonistic activity of organotin compounds on estrogen receptors (ERs), we examined the inhibitive effect of these compounds on estradiol-dependent beta-galactosidase activity using the yeast two-hybrid detection system consisting of a combination of the human estrogen receptor (hERbeta) ligand-binding domain and the co-activator steroid receptor co-activator-1 (SRC1). Tributyltin-hydroxide (TBT-OH) and triphenyltin-chlorine (TPT-Cl) exhibited an inhibitive effect on E₂-dependent transcriptional activity, similar to antagonistic chemicals such as 4-hydroxytamoxifen (OHT) or ICI 182,780, at a very low concentration of 10⁻¹⁴ M TBT or 10⁻¹⁰ M TPT, respectively. The yeast growth and transcriptional activity with transcriptional factor GAL4 did not exhibit any effect at the tested concentration of TBT or TPT. Moreover, the yeast two-hybrid system using the interaction between p53 and the T antigen of SV40 large did not describe any effect at the tested concentration of OHT or ICI 182,780. However, the interaction between p53 and T antigen was inhibited at a TBT or TPT concentration of 10⁻⁹ M, respectively. These results indicate that TBT and TPT act as inhibitors of ER-dependent reporter gene transcriptional activation and of the interaction between hERbeta LBD and the co-activator SRC1 in the yeast two-hybrid system. Consequently, our data could partly explain the occurrence of organotin compound-induced imposex on the endocrine system of mammals, including humans.

The term ‘sake yeast’ is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.

The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomyces cerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y. lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in β-oxidation and peroxisome proliferation by oleate was distinctly diminished in the Δpor1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica.

Phosphatidylethanolamine (PE) is one of the essential phospholipids in the yeast Saccharomyces cerevisiae. We have previously shown that a yeast strain, the endogenous PE synthesis of which was controllable, grew in the presence of PE containing decanoyl residues (diC10PE) when PE synthesis was repressed. In this study, we investigated the fate of diC10PE, its uptake and remodeling in yeast. Deletion of the genes encoding Lem3p/Ros3p or P-type ATPases, Dnf1p and Dnf2p, impaired the growth of the mutants in the medium containing diC10PE, suggesting the involvement of these proteins in the uptake of diC10PE. Analysis of the metabolism of deuterium-labeled diC10PE by electrospray ionization tandem mass spectrometry revealed that it was rapidly converted to deuterium-labeled PEs containing C16 or C18 acyl residues. The probable intermediate PEs that contained decanoic acid and C16 or C18 fatty acids as acyl residues were also detected. In addition, a substantial amount of decanoic acid was released into the culture medium during growth in the presence of diC10PE. These results imply that diC10PE was remodeled to PEs with longer acyl residues and used as membrane components. Defects in the remodeling of diC10PE in the deletion mutants of ALE1 and SLC1, products of which were capable of acyl-transfer to the sn− 2 position of lyso-phospholipids, suggested their involvement in the introduction of acyl residues to the sn− 2 position of lyso-phosphatidylethanolamine in the remodeling reaction of diC10PE. Our results also suggest the presence of a mechanism to maintain the physiological length of PE acyl residues in yeast.

A gene, CmCDR1, encoding an ABC transporter of the dicarboxylic acid (DCA)-producing yeast Candida maltosa was cloned. Transcription of CmCDR1 was upregulated in a DCA-hyper-producing mutant of C. maltosa in a later phase of culture on n-dodecane, but not in its parental strain. CmCDR1 expression was significantly induced by the longer-chain DCA in this mutant.

Chitin is one of the major cell wall components of ascomycete filamentous fungi, and chitin synthesis plays important roles in the morphogenesis of hyphae. In the Aspergillus nidulans genome, there are two genes, csmA and csmB, that encode a myosin motor-like domain (MMD) at their N-termini and a chitin synthase domain (CSD) at their C-termini. In our previous studies, we found that the MMD of CsmA was required for its functionality, and that CsmA and CsmB had certain overlapping functions essential for polarized filamentous growth. In this study, we constructed a strain that produced CsmB lacking the MMD (CSDeltaMB). This strain exhibited defects similar to those of the csmB deletion mutant. FLAG-tagged CSDeltaMB (CSDeltaMB-FLAG) did not properly localize to the hyphae. CsmB was co-immunoprecipitated with actin in vivo, whereas CSDeltaMB was not. These results suggest that the MMD of CsmB is crucial for its proper localization via interaction with actin.

In the alkane-assimilating yeast Yarrowia lipolytica, the expression of ALK1, a gene encoding cytochrome P450 that catalyzes the first step of n-alkane oxidation, is induced by n-alkanes. We previously demonstrated that two basic helix-loop-helix proteins, Yas1p and Yas2p, activate the transcription of ALK1 in an alkane-dependent manner by forming a heterocomplex and binding to alkane-responsive element 1 (ARE1), a cis-acting element in the ALK1 promoter. Here we identified an Opi1 family transcription factor, Yas3p, involved in the alkane-dependent transcription regulation of ALK genes. Deletion of YAS3 caused a significant increase in ALK1 mRNA in cells grown on glucose, glycerol, and n-alkanes. The YAS3 deletion also resulted in a marked elevation of reporter gene expression driven by an ARE1-containing promoter on glycerol and n-decane. Bacterially expressed Yas3p bound specifically to Yas2p, but not to Yas1p, in vitro. In addition, although green fluorescent protein-tagged Yas3p was localized in the nucleus in glucose-containing medium, it changed its localization to an endoplasmic reticulum-like compartment upon transfer to medium containing n-decane. These findings suggest that Yas3p functions as a master regulator of transcriptional response, which changes its localization between the nucleus and endoplasmic reticulum membrane in response to different carbon sources. Furthermore, quantitative real time PCR analysis of 12 ALK genes in YAS1, YAS2, and YAS3 deletion mutants suggested that Yas3p is involved in the transcriptional repression of a variety of ALK genes, including ALK1. In contrast, YAS3 deletion did not affect the mRNA level of an INO1 ortholog in Y. lipolytica, indicating functional diversity of Opi1 family transcription factors.

During the sporulation process of Saccharomyces cerevisiae, meiotic progression is accompanied by de novo formation of the prospore membrane inside the cell. However, it remains to be determined whether certain species of lipids are required for spore formation in yeast. In this study, we analyzed the requirement of the synthesis of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and ergosterol for spore formation using strains in which the synthesis of these lipids can be controlled. When synthesis of PE and PC was repressed, sporulation efficiency decreased. This suggests that synthesis of these phospholipids is vital to proper sporulation. In addition, sporulation was also impaired in cells with a lowered sterol content, raising the possibility that sterol content is also important for spore formation.

Disruption of an SCS2 ortholog impaired the growth of the alkane-assimilating yeast Yarrowia lipolytica on n-alkanes, particularly on n-decane, although the mRNA level of the ALK1 gene encoding a highly inducible cytochrome P450ALK was not much affected. The same disruption did not cause inositol auxotrophy, implying that Y. lipolytica SCS2 has a different function from its Saccharomyces cerevisiae counterpart.

In the long-chain dicarboxylic acids (DCA)-hyperproducing mutant Candida maltosa strains, methyl-ends of n-alkanes and fatty acids are hydroxylated by n-alkane inducible cytochromes P450 (P450alk), presumably as an essential step in DCA production. A significantly higher production of P450alks was observed in response to n-alkane in the DCA-hyperproducing mutant strain M2030 than in the wild-type strain 1098. Northern analysis demonstrated that n-tetradecane induction levels of mRNAs of all four ALK genes encoding major P450alk isoforms involved in n-alkane assimilation were significantly higher in the DCA-hyperproducing mutant than in the wild-type strain. Among these four ALK genes, enhancement of the transcriptional induction level of ALK5, which prefers fatty acids as substrates, was prominent in the mutant. In agreement with Northern analysis, promoters of ALK genes, especially that of ALK5, more strongly responded to n-alkanes in the DCA-hyperproducing mutant than in the wild-type strain. These results suggest that the transcriptional control of ALK genes in the DCA-hyperproducing mutant strains was altered preferably to accelerate DCA production.

The expression of the ALK1 gene, which encodes cytochrome P450, catalyzing the first step of alkane oxidation in the alkane-assimilating yeast Yarrowia lipolytica , is highly regulated and can be induced by alkanes. Previously, we identified a cis -acting element (alkane-responsive element 1 [ARE1]) in the ALK1 promoter. We showed that a basic helix-loop-helix (bHLH) protein, Yas1p, binds to ARE1 in vivo and mediates alkane-dependent transcription induction. Yas1p, however, does not bind to ARE1 by itself in vitro, suggesting that Yas1p requires another bHLH protein partner for its DNA binding, as many bHLH transcription factors function by forming heterodimers. To identify such a binding partner of Yas1p, here we screened open reading frames encoding proteins with the bHLH motif from the Y. lipolytica genome database and identified the YAS2 gene. The deletion of the YAS2 gene abolished the alkane-responsive induction of ALK1 transcription and the growth of the yeast on alkanes. We revealed that Yas2p has transactivation activity. Furthermore, Yas1p and Yas2p formed a protein complex that was required for the binding of these proteins to ARE1. These findings allow us to postulate a model in which bHLH transcription factors Yas1p and Yas2p form a heterocomplex and mediate the transcription induction in response to alkanes.

To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a Saccharomyces cerevisiae mutant deleted for ARE1 and ARE2 encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.

In the n-alkane-assimilating yeast Yarrowia lipolytica, the transcription of ALK1, encoding cytochrome P450, that catalyses n-alkane hydroxylation is activated by a complex composed of Yas1p and Yas2p via a promoter element, ARE1, in response to n-alkanes. An Opi1-family transcription factor, Yas3p, represses the transcription by binding to Yas2p in the nucleus when cultured in glucose-containing medium, but it is localized to the ER, presumably through interaction with acidic phospholipids, phosphatidic acid and/or phospho inositides, when cultured in n-alkane-containing medium. Here, to elucidate the mechanisms regulating the localization of Yas3p, point and deletion mutants of Yas3p were constructed and analysed. The substitution of Trp(360) and Cys(361) by Arg abrogated the localization of Yas3p to the ER and decreased ARE1-mediated transcriptional activation by n-alkane. A Yas3p truncation mutant consisting of residues 259-422 did not bind to acidic phospholipids, but it was localized to the ER in the presence of n-alkane, implying the acidic-phospholipid-independent recruitment of this mutant to the ER in response to n-alkane. The W360R and C361R substitutions in this truncation mutant abolished its localization to the ER. The results suggest that these residues are implicated in the acidic phospholipid-independent interaction of Yas3p to the ER.

The yeast Yarrowia lipolytica can assimilate hydrophobic substrates, including n-alkanes and fatty alcohols. Here, eight alcohol dehydrogenase genes, ADH1-ADH7 and FADH, and a fatty alcohol oxidase gene, FAO1, were analyzed to determine their roles in the metabolism of hydrophobic substrates. A mutant deleted for all of these genes (ALCY02 strain) showed severely defective growth on fatty alcohols, and enhanced sensitivity to fatty alcohols in glucose-containing media. The ALCY02 strain grew normally on n-tetradecane or n-hexadecane, but exhibited slightly defective growth on n-decane or n-dodecane. It accumulated more 1-dodecanol and less dodecanoic acid than the wild-type strain when n-dodecane was fed. Expression of ADH1, ADH3 or FAO1, but not that of other ADH genes or FADH, in the ALCY02 strain restored its growth on fatty alcohols. In addition, a triple deletion mutant of ADH1, ADH3 and FAO1 showed similarly defective growth on fatty alcohols and on n-dodecane to the ALCY02 strain. Microscopic observation suggests that Adh1p and Adh3p are localized in the cytosol and Fao1p is in the peroxisome. These results suggest that Adh1p, Adh3p and Fao1p are responsible for the oxidation of exogenous fatty alcohols but play less prominent roles in the oxidation of fatty alcohols derived from n-alkanes.

Cell wall formation and maintenance are crucial for hyphal morphogenesis. In many filamentous fungi, chitin is one of the main structural components of the cell wall. Aspergillus nidulans ChsB, a chitin synthase, and CsmA, a chitin synthase with a myosin motor-like domain (MMD) at its N-terminus, both localize predominantly at the hyphal tip regions and at forming septa. ChsB and CsmA play crucial roles in polarized hyphal growth in A. nidulans. In this study, we investigated the mechanism by which CsmA and ChsB accumulate at the hyphal tip in living hyphae. Deletion of kinA, a gene encoding conventional kinesin (kinesin-1), impaired the localization of GFP-CsmA and GFP-ChsB at the hyphal tips. The transport frequency of GFP-CsmA and GFP-ChsB in both anterograde and retrograde direction appeared lower in the kinA-deletion strain compared to wild type, although the velocities of the movements were comparable. Co-localization of GFP-ChsB and GFP-CsmA with mRFP1-KinArigor, a KinA mutant that binds to microtubules but does not move along them, was observed in the posterior of the hyphal tip regions. KinA co-immunoprecipitated with ChsB and CsmA. Co-localization and association of CsmA with KinA did not depend on the MMD. These findings indicate that ChsB and CsmA are transported along microtubules to the subapical region by KinA.

The polarized synthesis of cell wall components such as chitin is essential for the hyphal tip growth of filamentous fungi. The actin cytoskeleton is known to play important roles in the determination of hyphal polarity in Aspergillus nidulans. Previously, we suggested that CsmA, a chitin synthase with a myosin motor-like domain (MMD), was involved in polarized chitin synthesis in a manner dependent on the interaction between the MMD and the actin cytoskeleton. The genome database indicates that A. nidulans possesses another gene encoding another chitin synthase with an MMD. In this study, we characterized this gene, which we designated csmB. The csmB null mutants examined were viable, although they exhibited defective phenotypes, including the formation of balloons and intrahyphal hyphae and the lysis of subapical regions, which were similar to those obtained with csmA null mutants. Moreover, csmA csmB double null mutants were not viable. Mutants in which csmB was deleted and the expression of csmA was under the control of the alcA promoter were viable but severely impaired in terms of hyphal growth under alcA-repressing conditions. We revealed that CsmB with three copies of a FLAG epitope tag localized at the hyphal tips and forming septa, and that the MMD of CsmB was able to bind to actin filaments in vitro. These results suggest that CsmA and CsmB perform compensatory functions that are essential for hyphal tip growth.

Genome-wide screening has identified 37 Al-tolerance genes in Saccharomyces cerevisiae. These genes can be roughly categorised into three groups on the basis of function, i.e., genes related to vesicle transport processes, signal transduction pathways, and protein mannosylation. The largest group is composed of genes related to vesicle transport processes; severe Al sensitivity was found in yeast strains lacking these genes. The retrograde transport of endosome-derived vesicles back to the Golgi apparatus is an important factor in determining the Al tolerance of the vesicle transport system. The PKC1-MAPK cascade signalling pathway is important in the Al tolerance of signal transduction. The lack of the gene implicated in this process leads to weakened cell wall architecture, rendering the yeast Al-sensitive. Alternatively, Al might attack the cell wall and/or plasma membrane, and, as signalling is prevented in cells devoid of the genes related to signalling processes, the cells may be unable to alleviate the damage. The genes for protein mannosylation are also associated with Al tolerance, demonstrating the importance of cell wall architecture. These genes are involved in cell integrity processes.

The chsA and chsC encode classes II and I chitin synthases, respectively, of the filamentous fungus Aspergillus nidulans. The DeltachsA DeltachsC double mutants (DeltaAC mutants) show defects in asexual development: a striking reduction in the number of conidiophores and aberrant conidiophore morphology. Here, we examined the involvement of regulatory genes for asexual development (brlA, abaA, and medA) in the conidiation defects of the DeltaAC mutants. Spatial expression patterns of brlA, abaA, and medA in conidiophores of the wild-type strains and DeltaAC mutants were examined by in-situ staining using a reporter gene; expression of either gene was detected at abnormal sterigmata in the DeltaAC mutants as well as at normal ones in the wild-type strain. However, abaA expression was not prominent at a subset of conidiophores developing long chains of aberrant sterigmata, suggesting that induction of the abaA expression was retarded in the DeltaAC mutants. Based on these results and those previously presented, possible mechanisms involved in the conidiation defects are discussed.

The plaA gene encoding a protein that contains the cytosolic Phospholipase A(2) (cPLA(2)) motif is cloned for the first time from the filamentous fungus, Aspergillus nidulans. The translated 837 amino acid protein product of plaA comprises conserved lipase regions that are present in most mammalian cPLA(2) homologs. High expression of plaA was observed in glucose-lactose medium by Northern blot analyses. Deletion mutants of plaA grew and formed conidia similar to the wild-type strain, but showed decreased PLA(2) activity. Expression of the N-terminal truncated form of plaA in yeast cells resulted in increased Ca(2+)-dependent PLA(2) activity with (14)C-labeled phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as substrates, compared with vector-transformed cells. In conclusion, we have identified and cloned a phospholipid-hydrolyzing novel cPLA(2) protein from A. nidulans for the first time.

The ALK2 gene, encoding one of the n-alkane-hydroxylating cytochromes P450 in Candida maltosa, is induced by n-alkanes and a peroxisome proliferator, clofibrate. Deletion analysis of this gene's promoter revealed two cis-acting elements-an n-alkane-responsive element (ARE2) and a clofibrate-responsive element (CRE2)-that partly overlap in sequence but have distinct functions. ARE2-mediated activation responded to n-alkanes but not to clofibrate and was repressed by glucose. CRE2-mediated activation responded to polyunsaturated fatty acids and steroid hormones as well as to peroxisome proliferators but not to n-alkanes, and it was not repressed by glucose. Both elements mediated activation by oleic acid. Mutational analysis demonstrated that three CCG sequences in CRE2 were critical to the activation by clofibrate as well as to the in vitro binding of a specific protein to this element. These findings suggest that ALK2 is induced by peroxisome proliferators and steroid hormones through a specific CRE2-mediated regulatory mechanism.

One of the essential features of fungal morphogenesis is the polarized synthesis of cell wall components such as chitin. The actin cytoskeleton provides the structural basis for cell polarity in Aspergillus nidulans, as well as in most other eukaryotes. A class V chitin synthase, CsmA, which contains a myosin motor-like domain (MMD), is conserved among most filamentous fungi. The ΔcsmA null mutant showed remarkable abnormalities with respect to cell wall integrity and the establishment of polarity. In this study, we demonstrated that CsmA tagged with 9× HA epitopes localized near actin structures at the hyphal tips and septation sites and that its MMD was able to bind to actin. Characterization of mutants bearing a point mutation or deletion in the MMD suggests that the interaction between the MMD and actin is not only necessary for the proper localization of CsmA, but also for CsmA function. Thus, the finding of a direct interaction between the chitin synthase and the actin cytoskeleton provides new insight into the mechanisms of polarized cell wall synthesis and fungal morphogenesis.

chsA and chsC are genes encoding class II and I chitin synthases of Aspergillus nidulans respectively. In a previous study, chsA chsC double mutants showed various growth defects, suggesting that their cell wall architecture was disorganized and their cell wall integrity diminished. Here, we constructed chsA chsC chsD triple mutants and chsA chsC csmA triple mutants to investigate the role of the class IV and class V chitin synthases, ChsD and CsmA respectively, in maintaining the cell wall structure of the chsA chsC double mutant. The former triple mutant grew a little slower than the chsA chsC double mutant, but the two showed similar phenotypes. In contrast, the latter triple mutant exhibited severe growth defects, particularly under low osmotic conditions. The levels of the csmA transcript of the wild-type strain and chsA or chsC single mutants were markedly elevated under low osmotic conditions, while that of the chsA chsC double mutants was high even under such conditions. These and other results suggest that the function of csmA is important for the maintenance of cell wall integrity and the polarized growth of the chsA chsC double mutant.

Cell surface phosphatidylethanolamine (PE) of the yeast cell was probed by biotinylated Ro09-0198 (Bio-Ro), which specifically binds to PE and was visualized with fluorescein-labelled streptavidin. In Saccharomyces cerevisiae, the signals were observed at the presumptive bud site, the emerging small bud cortex, the bud neck of the late mitotic large-budded cells and the tip of the mating projection. In Schizosaccharomyces pombe, the signals were observed at one end or both ends of mono-nucleated cells and the division plane of the late mitotic cells. These sites were polarized ends in the yeast cells, implying that PE is exposed on the cell surface at cellular polarized ends. Treatment of S. cerevisiae cells with Ro09-0198 resulted in aberrant F-actin accumulation at the above sites, implying that limited surface exposure of PE is involved in the polarized organization of the actin cytoskeleton. Furthermore, S. cerevisiae ros3, dnf1 and dnf2 null mutants, which were known to be defective in the internalization of fluorescence-labelled PE, as well as the combinatorial mutants, were stained with Bio-Ro at the enlarging bud cortex, in addition to the Bio-Ro-staining sites of wild-type cells, suggesting that Ros3p, Dnf1p and Dnf2p are involved in the retrieval of exposed PE at the bud cortex.

When the alkane-assimilating yeast Yarrowia lipolytica is cultivated on n-alkanes, it changes cellular metabolism for adaptation by inducing cytochrome p450 and other genes. From a comparative analysis of promoters of alkane-inducible genes, we identified a cis-acting element, ARE1 (alkane responsive element 1), which provides transcription induction in response to n-alkanes. In a genetic selection for mutants that were defective in ARE1-mediated transcription induction in the presence of n-alkanes, we found that the YAS1 (yeast alkane signaling) gene is essential for alkane response. The YAS1 gene encodes a basic helix-loop-helix (bHLH) family protein. Loss of Yas1p causes defects in n-alkane-dependent transcription induction of the p450 gene and growth on n-alkanes. Yas1p localizes to nuclei and binds to promoters containing ARE1. Yas1p also binds to its own promoter, and the expression of YAS1 is induced by n-alkanes. These features suggest that Yas1p is a novel transcription factor mediating alkane signaling and that it provides an autoregulatory loop.

We cloned a gene pldA encoding a protein containing phospholipase D (PLD) motifs from a filamentous fungus Aspergillus nidulans. The deduced protein product of pldA consists of 833 amino acids and contains four conserved regions of a PLD gene family. Deletion mutants of pldA grew and formed conidia in a normal manner. Although PLD and transphosphatidylation activities against phosphatidylcholine of the mutant cell extract did not change, the Ca(2+)-dependent PLD activity against phosphatidylethanolamine was significantly reduced, but not in the wild-type cell extract. This activity was markedly enhanced by high osmotic growth conditions in the wild-type cells, and pldA of A. nidulans likely encodes a Ca(2+)-dependent phosphatidylethanolamine-hydrolyzing PLD.

A rapid downshift in the growth temperature of Saccharomyces cerevisiae from 30 to 10 degrees C resulted in an increase in transcript levels of the antioxidation genes SOD1 [encoding Cu-Zn superoxide dismutase (SOD)], CTT1 (encoding catalase T), and GSH1 (encoding gamma-glutamylcysteine synthetase). The cellular activities of SOD and catalase were also increased, indicating that the temperature downshift caused an antioxidant response. In support of this, a simultaneous increase in the intracellular level of H(2)O(2) was observed. The level of YAP1 mRNA, encoding a transcription factor critical for the oxidative stress response in this yeast, was also increased by the temperature downshift. However, deletion of YAP1 did not reduce the elevated mRNA levels of the antioxidant genes. This suggests that the temperature downshift-induced increase in the mRNA level of anti-oxidant genes is YAP1-independent.

The bph operon of Pseudomonas sp. KKS102 is constituted of 11 bph genes which encode enzymes for biphenyl assimilation. Growth of a mutant in which a large part of the bph operon was deleted was inhibited by biphenyl in a concentration-dependent manner. We constructed a series of bph operon deletion mutants and tested for their biphenyl sensitivity. Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bph operon and working in the early stage of the biphenyl degradation. The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl. These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bph operon.

To improve the capabilities of microorganisms relevant for biodegradation, we developed a new genetic approach and applied it to the bph operon ( bphEGF [orf4] A1A2A3CD [orf1] A4R ) of Pseudomonas sp. strain KKS102 to enhance its biphenyl- and polychlorinated biphenyl (PCB)-degrading activity. A native promoter of the bph operon, which was under control, was replaced through homologous recombination by a series of promoters that had constitutive activity. By testing a series of promoters with various strengths, we were able to obtain strains that have enhanced degradation activity for biphenyl and PCBs. This strategy removes the rate-limiting factor associated with transcription and has the potential to improve the degradation activity of a wide variety of microorganisms involved in biodegradation.

Ro09-0198 (Ro) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE) and causes cytolysis. To investigate the molecular basis of transbilayer movement of PE in biological membranes, we have isolated a series of budding yeast mutants that are hypersensitive to the Ro peptide. One of the most sensitive mutants, designated ros3 (Ro-sensitive 3), showed no significant change in the cellular phospholipid composition or in the sensitivity to amphotericin B, a sterol-binding polyene macrolide antibiotic. These results suggest that the mutation of ros3 affects the PE organization on the plasma membrane, rather than PE synthesis or overall organization of the membrane structures. By functional complementation screening, we identified the gene ROS3 affected in the mutant, and we showed that the hypersensitive phenotype was caused by the defective expression of the ROS3 gene product, Ros3p, an evolutionarily conserved protein with two putative transmembrane domains. Disruption of the ROS3 gene resulted in a marked decrease in the internalization of fluorescence-labeled analogs of PE and phosphatidylcholine, whereas the uptake of fluorescence-labeled phosphatidylserine and endocytic markers was not affected. Neither expression levels nor activities of ATP-binding cassette transporters of the ros3Delta cells differed from those of wild type cells, suggesting that Ros3p is not related to the multidrug resistance activities. Immunochemical analyses of the structure and subcellular localization showed that Ros3p was a glycosylated membrane protein localized in both the plasma membrane and the endoplasmic reticulum, and that a part of Ros3p was associated with the detergent-insoluble glycolipid-enriched complexes. These results indicate that Ros3p is a membrane glycoprotein that plays an important role in the phospholipid translocation across the plasma membrane.

Among the eight genes ( YlALK1 – YlALK8 ) encoding P450 cytochromes of the CYP52 family of the n -alkane-assimilating yeast Yarrowia lipolytica , Y1ALK1 is most highly induced by n -alkanes with short hydrocarbon chains, such as n -decane, and involved in the initial hydroxylation of n -alkane. To determine the factors regulating YlALK1 expression, we isolated an n -decane assimilation-deficient mutant, B0-6-1, whose YlALK1 expression level was lower than that of the wild-type. By complementation of the mutation of B0-6-1, we cloned a gene having an open reading frame of 1062 bp. The putative gene product is a protein of 354 amino acids and has significant homology to Pex10ps of other organisms. We named this gene YlPEX10 . YlPex10p has a C 3 HC 4 ring finger motif common among Pex10ps in its C-terminal region. This motif was also essential for the function of YlPex10p. Both B0-6-1 and a null mutant of YlPEX10 failed to form peroxisome and showed low-level transcription of YlALK1 after the change of carbon source to n -decane. Furthermore, YlPEX5 and YlPEX6 disruptants also showed low-level transcription of YlALK1 like the YlPEX10 disruptant and B0-6-1 mutant. We propose that in this organism peroxisome deficiency represses the expression of n -alkane-inducible YlALK1 encoding cytochrome P450ALK1.

The YlALK1 gene, which encodes cytochrome P450ALK1, plays a primary role in the assimilation of n-decane by yeast Yarrowia lipolytica and is inducible by n-decane at the transcriptional level. Deletion analysis of the YlALK1 promoter revealed that a 95-bp region on the YlALK1 promoter (from the position -400 to -304 upstream of the ATG codon) is essential for the induction by n-decane and we named this region ARR1 (alkane-responsive region). ARR1 was found to be made up of two different elements, ARE1 (alkane-responsive element 1; from -394 to -371) and ARE2 (from -325 to -305). By electrophoretic mobility shift assay, we found that the respective elements gave specific shift bands with the extracts from Y. lipolytica cells grown on n-alkane, but not much evidently from the cells grown on glycerol or glucose. This suggests that proteins that specifically bind to these elements are present and their binding or synthesis is dependent on n-alkane.