Involvement of lipid-translocating exporter family proteins in determination of myriocin sensitivity in budding yeast.

Myriocin is an inhibitor of serine palmitoyltransferase involved in the initial biosynthetic step for sphingolipids, and causes potent growth inhibition in eukaryotic cells. In budding yeast, Rsb1, Rta1, Pug1, and Ylr046c are known as the Lipid-Translocating Exporter (LTE) family and believed to contribute to export of various cytotoxic lipophilic compounds. It was reported that Rsb1 is a transporter responsible for export of intracellularly accumulated long-chain bases, which alleviate the cytotoxicity. In this study, it was found that LTE family genes are involved in determination of myriocin sensitivity in yeast. Analyses of effects of deletion and overexpression of LTE family genes suggested that all LTEs contribute to suppression of cytotoxicity of myriocin. It was confirmed that RSB1 overexpression suppressed reduction in complex sphingolipid levels caused by myriocin treatment, possibly exporting myriocin to outside of the cell. These results suggested that LTE family genes function as a defense mechanism against myriocin.

Identification of plb1 mutation that extends longevity via activating Sty1 MAPK in Schizosaccharomyces pombe.

To understand the lifespan of higher organisms, including humans, it is important to understand lifespan at the cellular level as a prerequisite. So, fission yeast is a good model organism for the study of lifespan. To identify the novel factors involved in longevity, we are conducting a large-scale screening of long-lived mutant strains that extend chronological lifespan (cell survival in the stationary phase) using fission yeast. One of the newly acquired long-lived mutant strains (No.98 mutant) was selected for analysis and found that the long-lived phenotype was due to a missense mutation (92Phe → Ile) in the plb1+ gene. plb1+ gene in fission yeast is a nonessential gene encoding a homolog of phospholipase B, but its functions under normal growth conditions, as well as phospholipase B activity, remain unresolved. Our analysis of the No.98 mutant revealed that the plb1 mutation reduces the integrity of the cellular membrane and cell wall and activates Sty1 via phosphorylation.

Simultaneous structural replacement of the sphingoid long-chain base and sterol in budding yeast.

The basic structures of membrane lipids that compose biomembranes differ among species; i.e., in mammals, the primary structure of long-chain base (LCB), the common backbone of ceramides and complex sphingolipids, is sphingosine, whereas, in yeast Saccharomyces cerevisiae, it is phytosphingosine, and S. cerevisiae does not have sphingosine. In addition, the sterol, which is coordinately involved in various functions with complex sphingolipids, is cholesterol in mammals, while in yeast it is ergosterol. Previously, it was found that yeast cells are viable when the structure of LCBs is replaced by sphingosine by supplying an exogenous LCB to cells lacking LCB biosynthesis. Here, we characterized yeast cells having sphingosine instead of phytosphingosine (sphingosine cells). Sphingosine cells exhibited a strong growth defect when biosynthesis of ceramides or complex sphingolipids was inhibited, indicating that, in the sphingosine cells, exogenously added sphingosine is required to be further metabolized. The sphingosine cells exhibited hypersensitivity to various environmental stresses and had abnormal plasma membrane and cell wall properties. Furthermore, we also established a method for simultaneous replacement of both LCB and sterol structures with those of mammals (sphingosine/cholesterol cells). The multiple stress hypersensitivity and abnormal plasma membrane and cell wall properties observed in sphingosine cells were also observed in sphingosine/cholesterol cells, suggesting that simultaneous replacement of both LCB and sterol structures with those of mammals cannot prevent these abnormal phenotypes. This is the first study to our knowledge showing that S. cerevisiae can grow even if LCB and sterol structures are simultaneously replaced with mammalian types.

Impaired biosynthesis of ergosterol confers resistance to complex sphingolipid biosynthesis inhibitor aureobasidin A in a PDR16-dependent manner.

Complex sphingolipids and sterols are coordinately involved in various cellular functions, e.g. the formation of lipid microdomains. Here we found that budding yeast exhibits resistance to an antifungal drug, aureobasidin A (AbA), an inhibitor of Aur1 catalyzing the synthesis of inositolphosphorylceramide, under impaired biosynthesis of ergosterol, which includes deletion of ERG6, ERG2, or ERG5 involved in the final stages of the ergosterol biosynthesis pathway or miconazole; however, these defects of ergosterol biosynthesis did not confer resistance against repression of expression of AUR1 by a tetracycline-regulatable promoter. The deletion of ERG6, which confers strong resistance to AbA, results in suppression of a reduction in complex sphingolipids and accumulation of ceramides on AbA treatment, indicating that the deletion reduces the effectiveness of AbA against in vivo Aur1 activity. Previously, we reported that a similar effect to AbA sensitivity was observed when PDR16 or PDR17 was overexpressed. It was found that the effect of the impaired biosynthesis of ergosterol on the AbA sensitivity is completely abolished on deletion of PDR16. In addition, an increase in the expression level of Pdr16 was observed on the deletion of ERG6. These results suggested that abnormal ergosterol biosynthesis confers resistance to AbA in a PDR16-dependent manner, implying a novel functional relationship between complex sphingolipids and ergosterol.

Loss of tolerance to multiple environmental stresses due to limitation of structural diversity of complex sphingolipids.

Structural diversity of complex sphingolipids is important for maintenance of various cellular functions; however, the overall picture of the significance of this structural diversity remains largely unknown. To investigate the physiological importance of the structural diversity of complex sphingolipids, we here constructed a complex sphingolipid structural diversity disruption library in budding yeast, which comprises 11 mutants including with combinations of deletions of sphingolipid-metabolizing enzyme genes. The sensitivity of the mutants to various environmental stresses revealed that the more the structural variation of complex sphingolipids is limited, the more stress sensitivity tends to increase. Moreover, it was found that in mutant cells with only one subtype of complex sphingolipid, Slt2 MAP kinase and Msn2/4 transcriptional factors are essential for maintenance of a normal growth and compensation for reduced tolerance of multiple stresses caused by loss of complex sphingolipid diversity. Slt2 and Msn2/4 are involved in compensation for impaired integrity of cell walls and plasma membranes caused by loss of complex sphingolipid diversity, respectively. From these findings, it was suggested that loss of structural diversity of complex sphingolipids affects the environment of the cell surface, including both plasma membranes and cell walls, which could cause multiple environmental stress hypersensitivity.

Involvement of the mitochondrial retrograde pathway in dihydrosphingosine-induced cytotoxicity in budding yeast.

Sphingoid long-chain bases are essential intermediates of ceramides and complex sphingolipids, and function in the regulation of various signal transduction systems. Previously, we found that, in budding yeast, intracellularly accumulated dihydrosphingosine (DHS) causes mitochondrial reactive-oxygen species (ROS)-mediated cytotoxicity, which is much stronger than phytosphingosine. In this study, we screened for suppressor mutations that confer resistance to DHS, and identified RTG2, which encodes upregulation of the mitochondrial retrograde signaling pathway (RTG pathway). Deletion of RTG3 encoding transcriptional factor for the RTG pathway suppressed the cytotoxicity of DHS, whereas deletion of MKS1 or point mutation of LST8, both of which cause increased activity of the RTG pathway, enhanced the cytotoxicity. Moreover, the deletion of RTG3 also suppressed the DHS-induced increases in ROS levels. Finally, it was found that the RTG pathway is activated on DHS treatment. These results suggested that the cytotoxicity of DHS is partially mediated through activation of the RTG pathway.

Regulation of sphingolipid biosynthesis in the endoplasmic reticulum via signals from the plasma membrane in budding yeast.

Saccharomyces cerevisiae LIP1 encodes a regulatory subunit that forms a complex with the ceramide synthase catalytic subunits, Lag1/Lac1, which is localized on the membrane of endoplasmic reticulum. To understand the underlying regulatory mechanism of sphingolipid biosynthesis, we generated strains upon replacing the chromosomal LIP1 promoter with a Tet-off promoter, which enables the expression in Dox-dependent manner. The lip1-1 strain, obtained through the promoter substitution, exhibits severe growth inhibition and remarkable decrease in sphingolipid synthesis in the presence of Dox. Using this strain, we investigated the effect of a decrease in ceramide synthesis on TOR complex 2 (TORC2)-Ypk1 signaling, which senses the complex sphingolipid level at the plasma membrane and promotes sphingolipid biosynthesis. In lip1-1 cells, Ypk1 was activated via both upstream kinases, TORC2 and yeast PDK1 homologues, Pkh1/2, thereby inducing hyperphosphorylation of Lag1, but not of another Ypk1-substrate, Orm1, which is a known negative regulator of the first step of sphingolipid metabolism, in the presence of Dox. Therefore, our data suggest that the metabolic enzyme activities at each step of the sphingolipid biosynthetic pathway are controlled through a fine regulatory mechanism.

Crosstalk between protein kinase A and the HOG pathway under impaired biosynthesis of complex sphingolipids in budding yeast.

Complex sphingolipids are important components of the lipid bilayer of budding yeast Saccharomyces cerevisiae, and a defect of the biosynthesis causes widespread cellular dysfunction. In this study, we found that mutations causing upregulation of the cAMP/protein kinase A (PKA) pathway cause hypersensitivity to the defect of complex sphingolipid biosynthesis caused by repression of AUR1 encoding inositol phosphorylceramide synthase, whereas loss of PKA confers resistance to the defect. Loss of PDE2 encoding cAMP phosphodiesterase or PKA did not affect the reduction in complex sphingolipid levels and ceramide accumulation caused by AUR1 repression, suggesting that the change in sensitivity to the AUR1 repression due to the mutation of the cAMP/PKA pathway is not caused by exacerbation or suppression of the abnormal metabolism of sphingolipids. We also identified PBS2 encoding MAPKK in the high-osmolarity glycerol (HOG) pathway as a multicopy suppressor gene that rescues the hypersensitivity to AUR1 repression caused by deletion of IRA2, which causes hyperactivation of the cAMP/PKA pathway. Since the HOG pathway has been identified as one of the rescue systems against the growth defect caused by the impaired biosynthesis of complex sphingolipids, it was assumed that PKA affects activation of the HOG pathway under AUR1-repressive conditions. Under AUR1-repressive conditions, hyperactivation of PKA suppressed the phosphorylation of Hog1, MAPK in the HOG pathway, and transcriptional activation downstream of the HOG pathway. These findings suggested that PKA is possibly involved in the avoidance of excessive activation of the HOG pathway under impaired biosynthesis of complex sphingolipids.

[Case Report of Four Patients Received Ramucirumab Monotherapy as Second-Line Chemotherapy for Advanced Gastric Cancer].

Ramucirumab monotherapy is one of the conditionally recommended regimens in second-line chemotherapy for advanced gastric cancer. However, there are few clinical data on ramucirumab monotherapy in Japanese patients. Herein, we present 4 case reports of advanced gastric cancer patients who received ramucirumab monotherapy. The 4 patients' age ranged from 65-81 years old(median: 70 years old), with a 3:1 male to female ratio. Since all cases were in poor performance status, administration of cell-killing anticancer drugs such as paclitaxel was contraindicated, and ramucirumab monotherapy was selected as an alternative. Ramucirumab was administrated 2-8 times(median: 3 times), resulting to a stable disease in 1 patient, and progression-free survival was noted to be 3-16 weeks(median: 5 weeks). Regarding complications, Grade 2 hypertension occurred in 1 patient, and no serious adverse events were observed. Ramucirumab monotherapy is a well-tolerated second-line chemotherapy for patients with advanced gastric cancer in poor performance status, and it is expected to have some disease control effect.

Identification of sur2 mutation affecting the lifespan of fission yeast.

Yeast is a suitable model system to analyze the mechanism of lifespan. In this study, to identify novel factors involved in chronological lifespan, we isolated a mutant with a long chronological lifespan and found a missense mutation in the sur2+ gene, which encodes a homolog of Saccharomyces cerevisiae sphingolipid C4-hydroxylase in fission yeast. Characterization of the mutant revealed that loss of sur2 function resulted in an extended chronological lifespan. The effect of caloric restriction, a well-known signal for extending lifespan, is thought to be dependent on the sur2+ gene.

ROS-mediated synthetic growth defect caused by impaired metabolism of sphingolipids and phosphatidylserine in budding yeast.

Previously, we found that yeast exhibits a strong growth defect with the combination of a lack of gene involved in structural modification of sphingolipids and repression of the phosphatidylserine synthase gene. Here we found that the double gene mutation causes reactive oxygen species-mediated cell growth defect, which is suppressed by deletion of LEM3 encoding the subunit of phospholipid flippase.

Proper regulation of inositolphosphorylceramide levels is required for acquirement of low pH resistance in budding yeast.

All organisms have stress response systems to protect themselves from various environmental stresses, and regulation of membrane lipids is thought to play an important role in acquirement of stress tolerance. Complex sphingolipids in the yeast Saccharomyces cerevisiae are classified into three types based on differences in the structure of the polar head group, and the compositions and quantities of complex sphingolipids in biomembranes are tightly regulated. In this study, we found that the accumulation of inositol phosphorylceramides (IPCs) due to a defect of mannosylinositol phosphorylceramide biosynthesis (sur1∆ csh1∆), i.e., disruption of the balance of the composition of complex sphingolipids, causes hypersensitivity to low pH conditions (pH 4.0-2.5). Furthermore, screening of suppressor mutations that confer low pH resistance to sur1∆ csh1∆ cells revealed that a change in ergosterol homeostasis at plasma membranes can rescue the hypersensitivity, suggesting the functional relationship between complex sphingolipids and ergosterol under low pH conditions. Under low pH conditions, wild-type yeast cells exhibited decreases in IPC levels, and forced enhancement of the biosynthesis of IPCs causes low pH hypersensitivity. Thus, it was suggested that the accumulation of IPCs is detrimental to yeast under low pH conditions, and downregulation of IPC levels is one of the adaptation mechanisms for low pH conditions.

Mitochondrial reactive oxygen species-mediated cytotoxicity of intracellularly accumulated dihydrosphingosine in the yeast Saccharomyces cerevisiae.

In eukaryotic cells, the content of sphingoid long-chain bases (LCBs) is generally much lower than that of complex sphingolipids and ceramides, and the quantitative balance of these metabolites in cells is tightly regulated. In the budding yeast Saccharomyces cerevisiae, it has been demonstrated that exogenously added phytosphingosine (PHS) causes a strong growth defect in tryptophan auxotrophic cells, due to delayed uptake of tryptophan from the culture medium; however, the growth inhibitory effect of dihydrosphingosine (DHS) is less than that of PHS in tryptophan auxotrophic cells. Here, we found that, in tryptophan-prototrophic yeast cells, exogenously added DHS is much more toxic than PHS. Exogenously added DHS is converted to PHS, Cers, or LCB 1-phosphates through the action of sphingolipid C4-hydroxylase, Cer synthases, or LCB kinases, respectively; however, suppression of further metabolism of DHS in cells resulted in an increase in the growth inhibitory activity of exogenously added DHS, indicating that DHS itself is causative of the cytotoxicity. The cytotoxicity of DHS was not mediated by Pkh1/2, Sch9, and Ypk1/2 kinases, intracellular targets of LCBs. DHS treatment caused an increase in mitochondria-derived reactive oxygen species, and the cytotoxic effect of DHS was suppressed by depletion of mitochondrial DNA or antioxidant N-acetylcysteine, but enhanced by deletion of SOD1 and SOD2 encoding superoxide dismutases. Thus, collectively, these results indicated that intracellularly accumulated DHS has mitochondrial reactive oxygen species-mediated cytotoxic activity, which is much more potent than that of PHS.

Mannosylinositol phosphorylceramides and ergosterol coodinately maintain cell wall integrity in the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, complex sphingolipids have three types of polar head group, and breakdown of their normal composition causes several cellular dysfunctions. Previously we found that loss of biosynthesis of mannosylinositol phosphorylceramide (MIPC) causes a defect in cell wall integrity (CWI). In this study, we screened for multicopy suppressor genes that rescue the defect in CWI in cells lacking MIPC synthases (Sur1 and Csh1), and found that the defect is partly suppressed by upregulation of ergosterol biosynthesis. In addition, repression of expression of ERG9, which encodes squalene synthase in the ergosterol biosynthesis pathway, in sur1∆ csh1∆ cells caused a strong growth defect and enhancement of the defect in CWI. The repression of ERG9 and/or the deletion of SUR1 and CSH1 caused an increase in the phosphorylated form of Slt2, a mitogen-activated protein kinase activated through impairment of CWI. Moreover, the deletion of SLT2 or WSC1/2, encoding a sensor protein recognizing CWI, enhanced the growth defect in the ERG9-repressed sur1∆ csh1∆ cells. On the other hand, the ERG9-repressed sur1∆ csh1∆ cells also exhibited an increase in the cell wall chitin level in a Slt2- and Wsc1/2-independent manner. These results suggested that MIPC and ergosterol are coordinately involved in maintenance of CWI, and the activation of Slt2 suppressed the CWI defect caused by these metabolic defects.

Protection mechanisms against aberrant metabolism of sphingolipids in budding yeast.

Life is dependent on the protection of cellular functions from various stresses. Sphingolipids are essential biomembrane components in eukaryotic organisms, which are exposed to risks that may disrupt sphingolipid metabolism, threatening their lives. Defects of the sphingolipid biosynthesis pathway cause profound defects of various cellular functions and ultimately cell death. Therefore, cells are equipped with defense response mechanisms against aberrant metabolism of sphingolipids, the most characterized one being the target of rapamycin complex 2-mediated regulation of sphingolipid biosynthesis in budding yeast Saccharomyces cerevisiae. On the other hand, very recently, we found that the high osmolarity glycerol pathway is involved in suppression of a growth defect caused by a reduction in complex sphingolipid levels in yeast. It is suggested that this signaling pathway is not involved in the repair of the impaired biosynthesis pathway for sphingolipids, but compensates for cellular dysfunctions caused by reduction in complex sphingolipid levels. This is a novel protection mechanism against aberrant metabolism of complex sphingolipids, and further investigation of the mechanism will provide new insights into the physiological significance of complex sphingolipids. Here, we summarize the response signaling against breakdown of sphingolipid biosynthesis in yeast, which includes the high osmolarity glycerol pathway.

Protective role of the HOG pathway against the growth defect caused by impaired biosynthesis of complex sphingolipids in yeast Saccharomyces cerevisiae.

Complex sphingolipids play critical roles in various cellular events in the yeast Saccharomyces cerevisiae. To identify genes that are related to the growth defect caused by disruption of complex sphingolipid biosynthesis, we screened for suppressor mutations and multicopy suppressor genes that confer resistance against repression of AUR1 encoding inositol phosphorylceramide synthase. From the results of this screening, we found that the activation of high-osmolarity glycerol (HOG) pathway is involved in suppression of growth defect caused by impaired biosynthesis of complex sphingolipids. Furthermore, it was found that transcriptional regulation via Msn2, Msn4 and Sko1 is involved in the suppressive effect of the HOG pathway. Lack of the HOG pathway did not enhance the reductions in complex sphingolipid levels or the increase in ceramide level caused by the AUR1 repression, implying that the suppressive effect of the HOG pathway on the growth defect is not attributed to restoration of impaired biosynthesis of complex sphingolipids. On the contrary, the HOG pathway and Msn2/4-mediated transcriptional activation was involved in suppression of aberrant reactive oxygen species accumulation caused by the AUR1 repression. These results indicated that the HOG pathway plays pivotal roles in maintaining cell growth under impaired biosynthesis of complex sphingolipids.

Overexpression of PDR16 confers resistance to complex sphingolipid biosynthesis inhibitor aureobasidin A in yeast Saccharomyces cerevisiae.

Sphingolipids are essential for normal cell growth of yeast Saccharomyces cerevisiae. Aureobasidin A (AbA), an antifungal drug, inhibits Aur1, an enzyme catalyzing the synthesis of inositol phosphorylceramide, and induces a strong growth defect in yeast. In this study, we screened for multicopy suppressor genes that confer resistance to AbA, and identified PDR16. In addition, it was found that PDR17, a paralog of PDR16, also functions as a multicopy suppressor. Pdr16 and Pdr17 belong to a family of phosphatidylinositol transfer proteins; however, cells overexpressing the other members of the family hardly exhibited resistance to AbA. Overexpression of a lipid-binding defective mutant of Pdr16 did not confer the resistance to AbA, indicating that the lipid-binding activity is essential for acquiring resistance to AbA. When expression of the AUR1 gene was repressed by a tetracycline-regulatable promoter, the overexpression of PDR16 or PDR17 did not suppress the growth defect caused by the AUR1 repression. Quantification analysis of complex sphingolipids revealed that in AbA-treated cells, but not in cells in which AUR1 was repressed by the tetracycline-regulatable promoter, the reductions of complex sphingolipid levels were suppressed by the overexpressed PDR16. Thus, it was indicated that the overexpression of PDR16 reduces the effectiveness of AbA against intracellular Aur1 activity.

Yeast lacking the amphiphysin family protein Rvs167 is sensitive to disruptions in sphingolipid levels.

Rvs167 and Rvs161 in Saccharomyces cerevisiae are amphiphysin family proteins, which are involved in several important cellular events, such as invagination and scission of endocytic vesicles, and actin cytoskeleton organization. It has been reported that cellular dysfunctions caused by deletion of RVS167 or RVS161 are rescued by deletion of specific nonessential sphingolipid-metabolizing enzyme genes. Here, we found that yeast cells lacking RVS167 or RVS161 exhibit a decrease in sphingolipid levels. In rvs167∆ cells, the expression level of Orm2, a negative regulator of serine palmitoyltransferase (SPT) catalyzing the initial step of sphingolipid biosynthesis, was increased in a calcineurin-dependent manner, and the decrease in sphingolipid levels in rvs167∆ cells was reversed on deletion of ORM2. Moreover, repression of both ORM1 and ORM2 expression or overexpression of SPT caused a strong growth defect of rvs167∆ cells, indicating that enhancement of de novo sphingolipid biosynthesis is detrimental to rvs167∆ cells. In contrast, partial repression of LCB1-encoding SPT suppressed abnormal phenotypes caused by the deletion of RVS167, including supersensitivity to high temperature and salt stress, and impairment of endocytosis and actin cytoskeleton organization. In addition, the partial repression of SPT activity suppressed the temperature supersensitivity and abnormal vacuolar morphology caused by deletion of VPS1 encoding a dynamin-like GTPase, which is required for vesicle scission and is functionally closely related to Rvs167/Rvs161, whereas repression of both ORM1 and ORM2 expression in vps1∆ cells caused a growth defect. Thus, it was suggested that proper regulation of SPT activity is indispensable for amphiphysin-deficient cells.

Phospholipid methylation controls Atg32-mediated mitophagy and Atg8 recycling.

Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy-inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria-anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8-PMME, a mitophagy-incompetent lipid conjugate of the autophagy-related ubiquitin-like modifier. Amelioration of Atg32 expression and attenuation of Atg8-PMME conjugation markedly rescue mitophagy in opi3-null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32-mediated mitophagy.

VID22 is required for transcriptional activation of the PSD2 gene in the yeast Saccharomyces cerevisiae.

Phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae is synthesized through decarboxylation of phosphatidylserine (PS), catalysed by PS decarboxylase 1 (Psd1p) and 2 (Psd2p) and the cytidine 5'-diphosphate (CDP)-ethanolamine (CDP-Etn) pathway. PSD1 null (psd1Δ) and PSD2 null (psd2Δ) mutants are viable in a synthetic minimal medium, but a psd1Δ psd2Δ double mutant exhibits Etn auxotrophy, which is incorporated into PE through the CDP-Etn pathway. We have previously shown that psd1Δ is synthetic lethal with deletion of VID22 (vid22Δ) [Kuroda et al. (2011) Mol. Microbiol. 80: , 248-265]. In the present study, we found that vid22Δ mutant exhibits Etn auxotrophy under PSD1-depressed conditions. Deletion of VID22 in wild-type and PSD1-depressed cells caused partial defects in PE formation through decarboxylation of PS. The enzyme activity of PS decarboxylase in an extract of vid22Δ cells was ∼70% of that in wild-type cells and similar to that in psd2Δ cells and the PS decarboxylase activity remaining in the PSD1-depressed cells became almost negligible with deletion of VID22. Thus, the vid22Δ mutation was suggested to cause a defect in the Psd2p activity. Furthermore, vid22Δ cells were shown to be defective in expression of the PSD2 gene tagged with 6×HA, the defect being ameliorated by replacement of the native promoter of the PSD2 gene with a CYC1 promoter. In addition, an α-galactosidase reporter assay revealed that the activity of the promoter of the PSD2 gene in vid22Δ cells was ∼5% of that in wild-type cells. These results showed that VID22 is required for transcriptional activation of the PSD2 gene.