Ryouichi Fukuda

Local exposure of phosphatidylethanolamine on the yeast plasma membrane is implicated in cell polarity.

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.

Growth temperature downshift induces antioxidant response in Saccharomyces cerevisiae.

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.

ACCUMULATION OF MISFOLDED PROTEIN AGGREGATES LEADS TO THE FORMATION OF RUSSELL BODY-LIKE DILATED ENDOPLASMIC RETICULUM IN YEAST

RNAP‐I, an aspartic proteinase from a filamentous fungus Rhizopus niveus, is secreted very efficiently in Saccharomyces cerevisiae. It is synthesized first as a precursor form with signal sequence and prosequence in its amino‐terminus. Our previous study indicated that the prosequence of RNAP‐I had important roles in its correct folding and secretion in yeast, and that a prosequence‐deleted derivative of RNAP‐I, Δpro, was not secreted but was retained and degraded in the yeast endoplasmic reticulum (ER). In the present study, we show that the accumulation of Δpro in the yeast ER caused elevated synthesis of ER resident chaperones, indicating that Δpro is recognized as an unfolded protein species in the ER. Our biochemical data demonstrated that Δpro formed aggregates which contained BiP, but not protein disulfide isomerase (PDI), in the ER. Immunoelectron microscopical analysis revealed that the Δpro aggregates were indeed visible as electron‐dense regions in the ER and nuclear envelope. Such ‘chaperone‐associated misfolded protein bodies’ were observed for the first time in yeast. Morphologies of the ER and nucleus were drastically altered by the accumulation of the Δpro aggregates. The ER lost its flat cisternal shape; the ER lumen extended aberrantly and the ER membrane irregularly proliferated. The misfolded Δpro proteins are probably sorted from the ordinary ER lumen to form the aggregates so that the ER function would not be grossly impaired, and the dilated ER may represent an ER subcompartment where the Δpro aggregates are degraded.

Basic Helix-Loop-Helix Transcription Factor Heterocomplex of Yas1p and Yas2p Regulates Cytochrome P450 Expression in Response to Alkanes in the Yeast Yarrowia lipolytica

ABSTRACT 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.

A basic helix-loop-helix transcription factor essential for cytochrome p450 induction in response to alkanes in yeast Yarrowia lipolytica.

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.

Yas3p, an Opi1 Family Transcription Factor, Regulates Cytochrome P450 Expression in Response to n-Alkanes in Yarrowia lipolytica

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.

Incorporation and remodeling of extracellular phosphatidylcholine with short acyl residues in Saccharomyces cerevisiae.

The pem1/cho2 pem2/opi3 double mutant of Saccharomyces cerevisiae, which is auxotrophic for choline because of the deficiency in methylation activities of phosphatidylethanolamine, grew in the presence of 0.1 mM dioctanoyl-phosphatidylcholine (diC(8)PC). Analysis of the metabolism of methyl-(13)C-labeled diC(8)PC ((methyl-(13)C)(3)-diC(8)PC) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) revealed that it was rapidly converted to (methyl-(13)C)(3)-PCs containing C16 or C18 acyl chains. (Methyl-(13)C)(3)-8:0-lyso-PC, (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC, which are the probable intermediate molecular species of acyl chain remodeling, appeared immediately after 5 min of pulse-labeling and decreased during the subsequent chase period. These results indicate that diC(8)PC was taken up by the pem1 pem2 double mutant and that the acyl chains of diC(8)PC were exchanged with longer yeast fatty acids. The temporary appearance of (methyl-(13)C)(3)-8:0-lyso-PC suggests that the remodeling reaction may consist of deacylation and reacylation by phospholipase activities and acyltransferase activities, respectively. The detailed analyses of the structures of (methyl-(13)C)(3)-8:0-16:0-PC and (methyl-(13)C)(3)-8:0-16:1-PC by MS/MS and MS(3) strongly suggest that most (methyl-(13)C)(3)-8:0-16:0-PCs have a C16:0 acyl chain at sn-1 position, whereas (methyl-(13)C)(3)-8:0-16:1-PCs have a C16:1 acyl chain at either sn-1 or sn-2 position in a similar frequency, implying that the initial C16:0 acyl chain substitution prefers the sn-1 position; however, the C16:1 acyl chain substitution starts at both sn-1 and sn-2 positions. The current study provides a pivotal insight into the acyl chain remodeling of phospholipids in yeast.

Genome-wide screening of aluminum tolerance in Saccharomyces cerevisiae

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.

Metabolism of Hydrophobic Carbon Sources and Regulation of It in n-Alkane-Assimilating Yeast Yarrowia lipolytica

A potent ability to assimilate hydrophobic compounds, including n-alkanes and fatty acids as carbon sources, is one of important characteristics of the yeast Yarrowia lipolytica, and has been studied for both basic microbiological interest and biotechnological applications. This review summarizes recent progress on the metabolism of n-alkanes and its transcriptional control in response to n-alkanes and to fatty acids in Y. lipolytica. In the metabolism of n-alkanes, cytochromes P450ALK catalyze their initial hydroxylation to fatty alcohols, which are subsequently converted to fatty acids and utilized. The transcription of ALK1, encoding a predominant cytochrome P450ALK, is regulated in response to n-alkanes by two basic helix-loop-helix transcription activators, Yas1p and Yas2p, and Opi1-family transcription repressor Yas3p. Transcription of the genes involved in fatty acid utilization and peroxisome biogenesis is controlled by Ctf1-family Zn2Cys6 type transcription factor Por1p in response to fatty acids in Y. lipolytica.

Construction and characterization of a Yarrowia lipolytica mutant lacking genes encoding cytochromes P450 subfamily 52.

The initial hydroxylation of n-alkane is catalyzed by cytochrome P450ALK of the CYP52 family in the n-alkane-assimilating yeast Yarrowia lipolytica. A mutant with a deletion of all 12 genes, ALK1 to ALK12, which are deduced to encode cytochromes P450 of the CYP52 family in Y. lipolytica, was successfully constructed. This deletion mutant, Δalk1-12, completely lost the ability to grow on n-alkanes of 10-16 carbons. In contrast, Δalk1-12 grew on the metabolite of n-dodecane, i.e., n-dodecanol, n-dodecanal, or n-dodecanoic acid, as well as the wild-type strain. In addition, production of n-dodecanoic acid was not observed when Δalk1-12 was incubated in the presence of n-dodecane. These results indicate the essential roles of P450ALKs in the oxidation of n-alkane. Δalk1-12 will be valuable as a host strain to express an individual ALK gene to elucidate the molecular function and substrate specificity of each P450ALK. Transcriptional activation of the ALK1 promoter by n-alkanes was observed in Δalk1-12 as in the wild-type strain, implying that n-alkanes per se, but not their metabolites, trigger n-alkane-induced transcriptional activation in Y. lipolytica.