Elucidating the functional role of human ABHD16B lipase in regulating triacylglycerol mobilization and membrane lipid synthesis in Saccharomyces cerevisiae.

Lipids are essential biological macromolecules that play a pivotal role in various physiological processes and cellular homeostasis. ABHD16B, a member of the α/ß-hydrolase domain (ABHD) superfamily protein, has emerged as a potential key regulator in lipid metabolism. However, the precise role of human ABHD16B in lipid metabolism remains unclear. In this study, we reported the overexpression of ABHD16B in Saccharomyces cerevisiae to determine its physiological relevance in lipid metabolism. Through in vivo [14C]acetate labeling experiments, we observed that overexpression of ABHD16B causes a decrease in cellular triacylglycerol (TAG) levels and a concurrent increase in phospholipid synthesis in wild-type cells. Mass spectrometry (LC-MS/MS) analysis further corroborated these findings, showing a significant decrease in TAGs with a carbon chain length of 48 and an increase in major phospholipid species, specifically 34:2, upon overexpression of ABHD16B. Confocal microscopy analysis revealed a reduction in the number of lipid droplets in strains overexpressing ABHD16B, consistent with the observed decrease in neutral lipids. Additionally, qRT-PCR analysis indicated a high phospholipid synthetic activity of ABHD16B and a potential decrease in TAG levels in wild-type yeast, possibly due to upregulation of endogenous TAG hydrolytic enzymes, as confirmed using 3tglsΔ mutant strain. Furthermore, GC-MS analysis revealed significant modifications in fatty acid composition upon ABHD16B overexpression. Collectively, our results underscore the influence of ABHD16B overexpression on TAG levels, phospholipid synthesis, lipid droplet dynamics, and fatty acid composition. These findings reveal a complex interplay between TAG hydrolysis and phospholipid synthesis, highlighting the critical involvement of ABHD16B in lipid homeostasis and providing further insights into its regulatory function in cellular lipid metabolism.

Molecular insights on PS-PLA1 lipase activity of human ABHD16B.

The human alpha beta hydrolase domain (ABHD) proteins are ubiquitous and regulate the cellular lipids' anabolic and catabolic processes. The structural aspects for specific biochemical function of many ABHD proteins related to physiological disorders and its link to pathological conditions remain unknown. Here putative human ABHD16B protein was overexpressed in Saccharomyces cerevisiae for its biological activity. In-vitro enzymatic assay of the recombinant ABHD16B protein with fluorescently tagged glycerophospholipids revealed that the PLA1 activity is observed with phosphatidylserine (PS). In addition, it efficiently hydrolyzed monoacylglycerol over triacylglycerols. Further, molecular dynamic simulations and per residue binding free energy decomposition analysis revealed that the origin of PS-specific PLA1 activity of ABHD16B is due to the electrostatic interaction of the PS head group with K8, R319, and E178, which led to having the hydrogen bond interaction of sn-1 acyl chain ester to the catalytic site residues. Site-directed mutagenesis of the 245GXSXG249 motif of ABHD16B reduced the maximal lipase activity of PS and MAG. In summary, these results revealed that ABHD16B plays a vital role in PS selectivity that in turn, controls the specific subcellular pools of 2-LPS metabolism in the tissues at low pH.

Impairment of transcription factor Gcr1p binding motif perturbs OPI3 transcription in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the transcription factor GCR1 plays a vital role in carbohydrate metabolism and in the current study we tried to elucidate its role in lipid metabolism. In silico analysis revealed the upstream activation sequence (UAS) in the promoter region of OPI3 possessed six conserved recognition sequences for Gcr1p and the ChIP assay confirmed the binding of Gcr1p on the OPI3 promoter region. The real-time quantitative polymerase chain reaction and promoter-reporter activity revealed a substantial reduction in OPI3 expression and was supported with decreased phosphatidylcholine (PC) level that is rescued with exogenous choline supplementation in gcr1∆ cells. Simultaneously, there was an increase in triacylglycerol level, accompanied with increased number and size of lipid droplets in gcr1∆ cells. The expression of pT1, pT2 truncations in opi3∆ cells revealed the -1 to -500 bp in the promoter region is essential for the activation of OPI3 transcription. The mutation specifically at UASCT box (-265) in the OPI3 promoter region displayed a reduction in the PC level and the additional mutation at UASINO (-165) further reduced the PC level. Collectively, our data suggest that the GCR1 transcription factor also regulates the OPI3 expression and has an impact on lipid homeostasis.

Activity-based protein profiling of rice (Oryza sativa L.) bran serine hydrolases.

Rice bran is an underutilized agricultural by-product with economic importance. The unique phytochemicals and fatty acid compositions of bran have been targeted for nutraceutical development. The endogenous lipases and hydrolases are responsible for the rapid deterioration of rice bran. Hence, we attempted to provide the first comprehensive profiling of active serine hydrolases (SHs) present in rice bran proteome by activity-based protein profiling (ABPP) strategy. The active site-directed fluorophosphonate probe (rhodamine and biotin-conjugated) was used for the detection and identification of active SHs. ABPP revealed 55 uncharacterized active-SHs and are representing five different known enzyme families. Based on motif and domain analyses, one of the uncharacterized and miss annotated SHs (Os12Ssp, storage protein) was selected for biochemical characterization by overexpressing in yeast. The purified recombinant protein authenticated the serine protease activity in time and protein-dependent studies. Os12Ssp exhibited the maximum activity at a pH between 7.0 and 8.0. The protease activity was inhibited by the covalent serine protease inhibitor, which suggests that the ABPP approach is indeed reliable than the sequence-based annotations. Collectively, the comprehensive knowledge generated from this study would be useful in expanding the current understanding of rice bran SHs and paves the way for better utilization/stabilization of rice bran.

A bioactive polypeptide from sugarcane selectively inhibits intestinal sucrase.

Human sucrase enzyme is a key therapeutic target for type 2 diabetes. While sugarcane sucrase inhibitor (sucinh) modulates invertase activity thereby accumulates sucrose. Molecular level understanding of sucinh towards mammalian α-glucosidases is scarce. The interaction of sucinh with human sucrase was identified and the association of these proteins was confirmed using co-purification, co-immunoprecipitation and pull-down assay. In addition, microscale thermophoresis assay showed that sucinh has a tight binding with sucrase (Kd = 4.77 nM) and a better affinity over acarbose. Collectively, in vitro, ex vivo and in silico data revealed that sucinh is selective for intestinal sucrase. The M region (H5/6 loop) of sucinh identified at the protein-protein interface is shown to have high affinity over N and C regions. Whereas, the biolayer luminescent imaging and microscale thermophoresis on the synthetic peptide of 28 amino acids of M region has a weak dose-dependent binding with sucrase. However, the synthetic peptide did not show substantial inhibition of sucrase and amylase activities at low concentration. Naturally derived carbohydrate mimics were shown to have a positive impact at the in vitro conditions. The insights obtained in this study give clues towards a new class of bioactive therapeutic peptides for α-glucosidases. A new horizon towards polypeptides derived from food sources emerge as a promising strategy for dietary interventions for prediabetic conditions.

Sesaminol diglucoside, a water-soluble lignan from sesame seeds induces brown fat thermogenesis in mice.

Brown adipose tissue (BAT) is the site of non-shivering thermogenesis in mammals, wherein energy is dissipated as heat. We observed that aqueous extract of black sesame seed triggers an increase in the expression of Uncoupling Protein 1 (UCP1) in brown adipocytes from mice. The active component from the extract was purified and identified to be sesaminol diglucoside (SDG). SDG treatment decreased mass of white fat pads and serum glucose levels and increased UCP1 levels in BAT thereby protecting mice against high fat induced weight gain. Further in silico and in vitro studies revealed that these effects are due to the agonist like behaviour of SDG towards beta 3 adrenergic receptors (ß3-AR). Together, our results suggest that SDG induces BAT mediated thermogenesis through ß3-AR and protects mice against diet-induced obesity.

Leaf lipidome and transcriptome profiling of Portulaca oleracea: characterization of lysophosphatidylcholine acyltransferase.

MAIN CONCLUSION: Portulaca leaves serve as an alternative bioresource for edible PUFAs. Transcriptome data provide information to explore Portulaca as a model system for galactolipids, leaf lipid metabolism, and PUFA-rich designer lipids. Poly-unsaturated fatty acids (PUFAs) are gaining importance due to their innumerable health benefits, and hence, understanding their biosynthesis in plants has attained prominence in recent years. The most common source of PUFAs is of marine origin. Although reports have identified Portulaca oleracea (purslane) as a leaf source of omega-3 fatty acids in the form of alpha-linolenic acid (ALA), the mechanism of ALA accumulation and its distribution into various lipids has not been elucidated. Here, we present the lipid profiles of leaves and seeds of several accessions of P. oleracea. Among the nineteen distinct accessions, the RR04 accession has the highest amount of ALA and is primarily associated with galactolipids. In addition, we report the transcriptome of RR04, and we have mapped the potential genes involved in lipid metabolism. Phosphatidylcholine (PC) is the major site of acyl editing, which is catalyzed by lysophosphatidylcholine acyltransferase (LPCAT), an integral membrane protein that plays a major role in supplying oleate to the PC pool for further unsaturation. Our investigations using mass spectrometric analysis of leaf microsomal fractions identified LPCAT as part of a membrane protein complex. Both native and recombinant LPCAT showed strong acyltransferase activity with various acyl-CoA substrates. Altogether, the results suggest that ALA-rich glycerolipid biosynthetic machinery is highly active in nutritionally important Portulaca leaves. Furthermore, lipidome, transcriptome, and mass spectrometric analyses of RR04 provide novel information for exploring Portulaca as a potential resource and a model system for studying leaf lipid metabolism.

Cell size is regulated by phospholipids and not by storage lipids in Saccharomyces cerevisiae.

Cell size and morphology are key adaptive features that influence almost all aspects of cellular physiology such as cell cycle and lipid metabolism. Here we report the role of a transcription factor Suppressor Phenotype of Ty elements insertion 10 (SPT10) of Saccharomyces cerevisiae in regulating cell cycle, cell size and lipid metabolism in concert, in addition to its defined role of histone gene expression. Morphological and biochemical analyses of spt10Δ strain show an abnormal cell size, cell cycle and lipid levels. The expression of Spt10p in spt10Δ strain helps the cell revert to typical wild-type phenotypes. SPT10 controls lipid metabolism by negatively regulating the expression of lipid biosynthetic genes, and positively regulating the expression of the lipid hydrolyzing genes. Spt10p helps in maintaining the cell size by regulating the amount of carbon flux into the phospholipid constituents of the cell membranes. On the contrary, storage lipids have no role in regulating the cell size. An exogenous supply of phosphatidic acid increases the cell size, proving the positive impact of the phospholipids on cell size modulation. SPT10 affects cell cycle, cell size and lipid metabolism by an orchestrated transcriptional regulation of the corresponding genes.

Arabidopsis serine/threonine/tyrosine protein kinase phosphorylates oil body proteins that regulate oil content in the seeds.

Protein phosphorylation is an important post-translational modification that can regulate the protein function. The current knowledge on the phosphorylation status of plant oil body (OB) proteins is inadequate. This present study identifies the distinct physiological substrates of Arabidopsis serine/threonine/tyrosine protein kinase (STYK) and its role in seed oil accumulation; the role of Arabidopsis OLE1, a major seed OB protein has also been elucidated. In vitro kinase assay followed by mass spectrometry identifies residue that are phosphorylated by STYK. Further, co-expression of OLE1 and STYK in yeast cells increases the cellular lipid levels and reduces the total lipid when OLE1 was replaced with OLE1T166A. Moreover, in vivo experiments with OB isolated from wild-type and styk knock-out lines show the ability of STYK to phosphorylate distinct OB proteins. OLE1T166A mutant and Arabidopsis styk mutant demonstrate the significant reduction of its substrate phosphorylation. styk mutant line significantly reduces the amount of total seed oil as compared to wild-type seeds. Together, our results provide the evidences that Arabidopsis At2G24360 (STYK) is phosphorylating oil body proteins and the phosphorylation regulates the oil content in Arabidopsis seeds.

The m6A methyltransferase Ime4 and mitochondrial functions in yeast.

In eukaryotes, the precise transcriptional and post-transcriptional regulations of gene expression are crucial for the developmental processes. More than 100 types of post-transcriptional RNA modifications have been identified in eukaryotes. The deposition of N6-methyladenosine (m6A) into mRNA is among the most common post-transcriptional RNA modifications known in eukaryotes. It has been reported that m6A RNA modification can regulate gene expression. The role of yeast m6A methyltransferase (Ime4) in meiosis and sporulation in diploid cells is very well proven, but its physiological role in haploid cells has remained unknown until recently. Previously, we have shown that Ime4 epitranscriptionally regulates triacylglycerol (TAG) metabolism and vacuolar morphology in haploid cells. Mitochondrial dysfunction leads to TAG accumulation as lipid droplets (LDs) in the cells; besides, LDs are physically connected to the mitochondria. As of now there are no reports on the role of Ime4 in mitochondrial biology. Here we report the important role played by Ime4 in the mitochondrial morphology and functions in Saccharomyces cerevisiae. The confocal microscopic analysis showed that IME4 gene deletion causes mitochondrial fragmentation; besides, the ime4Δ cells showed a significant decrease in cytochrome c oxidase and citrate synthase activities compared to the wild-type cells. IME4 gene deletion causes mitochondrial dysfunction, and it will be interesting to find out the target genes of Ime4 related to the mitochondrial biology. The determination of the role of Ime4 and its targets in mitochondrial biology could probably help in formulating potential cures for the mitochondria-linked rare genetic disorders.

The role of yeast m6A methyltransferase in peroxisomal fatty acid oxidation.

The precise and controlled regulation of gene expression at transcriptional and post-transcriptional levels is crucial for the eukaryotic cell survival and functions. In eukaryotes, more than 100 types of post-transcriptional RNA modifications have been identified. The N6-methyladenosine (m6A) modification in mRNA is among the most common post-transcriptional RNA modifications known in eukaryotic organisms, and the m6A RNA modification can regulate gene expression. The role of yeast m6A methyltransferase (Ime4) in meiosis, sporulation, triacylglycerol metabolism, vacuolar morphology, and mitochondrial functions has been reported. Stress triggers triacylglycerol accumulation as lipid droplets. Lipid droplets are physically connected to the different organelles such as endoplasmic reticulum, mitochondria, and peroxisomes. However, the physiological relevance of these physical interactions remains poorly understood. In yeast, peroxisome is the sole site of fatty acid ß-oxidation. The metabolic status of the cell readily governs the number and physiological function of peroxisomes. Under low-glucose or stationary-phase conditions, peroxisome biogenesis and proliferation increase in the cells. Therefore, we hypothesized a possible role of Ime4 in the peroxisomal functions. There is no report on the role of Ime4 in peroxisomal biology. Here, we report that IME4 gene deletion causes peroxisomal dysfunction under stationary-phase conditions in Saccharomyces cerevisiae; besides, the ime4Δ cells showed a significant decrease in the expression of the key genes involved in peroxisomal ß-oxidation compared to the wild-type cells. Therefore, identification and determination of the target genes of Ime4 that are directly involved in the peroxisomal biogenesis, morphology, and functions will pave the way to better understand the role of m6A methylation in peroxisomal biology.

The stress-regulatory transcription factors Msn2 and Msn4 regulate fatty acid oxidation in budding yeast.

The transcription factors Msn2 and Msn4 (multicopy suppressor of SNF1 mutation proteins 2 and 4) bind the stress-response element in gene promoters in the yeast Saccharomyces cerevisiae However, the roles of Msn2/4 in primary metabolic pathways such as fatty acid ß-oxidation are unclear. Here, in silico analysis revealed that the promoters of most genes involved in the biogenesis, function, and regulation of the peroxisome contain Msn2/4-binding sites. We also found that transcript levels of MSN2/MSN4 are increased in glucose-depletion conditions and that during growth in nonpreferred carbon sources, Msn2 is constantly localized to the nucleus in wild-type cells. Of note, the double mutant msn2Δmsn4Δ exhibited a severe growth defect when grown with oleic acid as the sole carbon source and had reduced transcript levels of major ß-oxidation genes. ChIP indicated that Msn2 has increased occupancy on the promoters of ß-oxidation genes in glucose-depleted conditions, and in vivo reporter gene analysis indicated reduced expression of these genes in msn2Δmsn4Δ cells. Moreover, mobility shift assays revealed that Msn4 binds ß-oxidation gene promoters. Immunofluorescence microscopy with anti-peroxisome membrane protein antibodies disclosed that the msn2Δmsn4Δ strain had fewer peroxisomes than the wild type, and lipid analysis indicated that the msn2Δmsn4Δ strain had increased triacylglycerol and steryl ester levels. Collectively, our data suggest that Msn2/Msn4 transcription factors activate expression of the genes involved in fatty acid oxidation. Because glucose sensing, signaling, and fatty acid ß-oxidation pathways are evolutionarily conserved throughout eukaryotes, the msn2Δmsn4Δ strain could therefore be a good model system for further study of these critical processes.

Unravelling a stearidonic acid-rich triacylglycerol biosynthetic pathway in the developing seeds of Buglossoides arvensis: A transcriptomic landscape.

Buglossoides arvensis is an emerging oilseed crop that is rich in stearidonic acid (SDA) and has several potential applications in human health and nutrition. The molecular basis of SDA biosynthesis in this plant remains unknown due to lack of genomic information. To unravel key genes involved in SDA-rich triacylglycerol (TAG) biosynthesis, we performed transcriptome sequencing of pooled mRNA from five different developmental stages of B. arvensis seeds using Illumina NextSeq platform. De novo transcriptome assembly generated 102,888 clustered transcripts from 39.83 million high-quality reads. Of these, 62.1% and 55.54% of transcripts were functionally annotated using Uniprot-Viridiplantae and KOG databases, respectively. A total of 10,021 SSR-containing sequences were identified using the MISA tool. Deep mining of transcriptome assembly using in silico tools led to the identification of genes involved in fatty acid and TAG biosynthesis. Expression profiling of 17 key transcripts involved in fatty acid desaturation and TAG biosynthesis showed expression patterns specific to the development stage that positively correlated with polyunsaturated fatty acid accumulation in the developing seeds. This first comprehensive transcriptome analysis provides the basis for future research on understanding molecular mechanisms of SDA-rich TAG accumulation in B. arvensis and aids in biotechnological production of SDA in other oilseed crops.

The m6A methyltransferase Ime4 epitranscriptionally regulates triacylglycerol metabolism and vacuolar morphology in haploid yeast cells.

N6-Methyladenosine (m6A) is among the most common modifications in eukaryotic mRNA. The role of yeast m6A methyltransferase, Ime4, in meiosis and sporulation in diploid strains is very well studied, but its role in haploid strains has remained unknown. Here, with the help of an immunoblotting strategy and Ime4-GFP protein localization studies, we establish the physiological role of Ime4 in haploid cells. Our data showed that Ime4 epitranscriptionally regulates triacylglycerol metabolism and vacuolar morphology through the long-chain fatty acyl-CoA synthetase Faa1, independently of the RNA methylation complex (MIS complex). The MIS complex consists of the Ime4, Mum2, and Slz1 proteins. Our affinity enrichment strategy (methylated RNA immunoprecipitation assays) using m6A polyclonal antibodies coupled with mRNA isolation, quantitative real-time PCR, and standard PCR analyses confirmed the presence of m6A-modified FAA1 transcripts in haploid yeast cells. The term "epitranscriptional regulation" encompasses the RNA modification-mediated regulation of genes. Moreover, we demonstrate that the Aft2 transcription factor up-regulates FAA1 expression. Because the m6A methylation machinery is fundamentally conserved throughout eukaryotes, our findings will help advance the rapidly emerging field of RNA epitranscriptomics. The metabolic link identified here between m6A methylation and triacylglycerol metabolism via the Ime4 protein provides new insights into lipid metabolism and the pathophysiology of lipid-related metabolic disorders, such as obesity. Because the yeast vacuole is an analogue of the mammalian lysosome, our findings pave the way to better understand the role of m6A methylation in lysosome-related functions and diseases.

Effect of zinc deprivation on the lipid metabolism of budding yeast.

Zinc is an essential micronutrient for all living cells. It serves as a structural and catalytic cofactor for numerous proteins, hence maintaining a proper level of cellular zinc is essential for normal functioning of the cell. Zinc homeostasis is sustained through various ways under severe zinc-deficient conditions. Zinc-dependent proteins play an important role in biological systems and limitation of zinc causes a drastic change in their expression. In budding yeast, a zinc-responsive transcription factor Zap1p controls the expression of genes required for uptake and mobilization of zinc under zinc-limiting conditions. It also regulates the polar lipid levels under zinc-limiting conditions to maintain membrane integrity. Deletion of ZAP1 causes an increase in triacylglyerol levels which is due to the increased biosynthesis of acetate that serves as a precursor for triacylglycerol biosynthesis. In this review, we expanded our recent work role of Zap1p in nonpolar lipid metabolism of budding yeast.

Human alpha beta hydrolase domain containing protein 11 and its yeast homolog are lipid hydrolases.

Mammalian alpha/beta hydrolase domain (ABHD) family of proteins have emerged as key regulators of lipid metabolism and are found to be associated with human diseases. Human α/ß-hydrolase domain containing protein 11 (ABHD11) has recently been predicted as a potential biomarker for human lung adenocarcinoma. In silico analyses of the ABHD11 protein sequence revealed the presence of a conserved lipase motif GXSXG. However, the role of ABHD11 in lipid metabolism is not known. To understand the biological function of ABHD11, we heterologously expressed the human ABHD11 in budding yeast, Saccharomyces cerevisiae. In vivo [14C]acetate labeling of cellular lipids in yeast cells overexpressing ABHD11 showed a decrease in triacylglycerol content. Overexpression of ABHD11 also alters the molecular species of triacylglycerol in yeast. Similar activity was observed in its yeast homolog, Ygr031w. The role of the conserved lipase motif in the hydrolase activity was proven by the mutation of all conserved amino acid residues of GXSXG motif. Collectively, our results demonstrate that human ABHD11 and its yeast homolog YGR031W have a pivotal role in the lipid metabolism.

Cardiolipin deficiency causes triacylglycerol accumulation in Saccharomyces cerevisiae.

In yeast, the synthesis of cardiolipin (CL) and phosphatidylethanolamine (PE) occurs mainly in mitochondria. CL and PE have overlapping functions, and they are required for mitochondrial function. PE is physiologically linked with triacylglycerol (TAG) metabolism in Saccharomyces cerevisiae, involving an acyl-CoA-independent pathway through the phospholipid:diacylglycerol acyltransferase activity of the Lro1 protein. There is no report on the physiological link between CL and TAG metabolism. Here we report a metabolic link between CL and TAG accumulation in the S. cerevisiae. Our data indicated that CL deficiency causes TAG accumulation, involving an acyl-CoA-dependent pathway through the diacylglycerol acyltransferase activity of the Dga1 protein with no changes in the TAG molecular species. The DGA1 gene deletion from the CL-deficient strains reduced the TAG levels. Data from in vitro and in vivo analyses showed that CL did not affect the enzymatic activity of Dga1. Our data also showed that CL deficiency leads to the up-regulation of acetyl-CoA synthetase genes (ACS1 and ACS2) of the cytosolic pyruvate dehydrogenase bypass pathway. This study establishes a physiological link between CL and TAG metabolism in S. cerevisiae.

The RNA polymerase I subunit Rpa12p interacts with the stress-responsive transcription factor Msn4p to regulate lipid metabolism in budding yeast.

In Saccharomyces cerevisiae, RPA12 encodes the small subunit of RNA polymerase I. Here, we demonstrate that Rpa12p interacts with the transcription factor Msn4p and prevents its binding to the promoter of AYR1 encoding Ayr1p (1-acyldihydroxyacetone phosphate reductase), a key enzyme involved in triacylglycerol biosynthesis and mobilization of nonpolar lipids. Deletion of RPA12 leads to triacylglycerol accumulation due to the binding of Msn4p to the promoter of AYR1 and activation of its transcription. The double deletion rpa12Δ::ayr1Δ caused a reduction in triacylglycerol levels. Our findings reveal that Rpa12p functions as a negative regulator of lipid metabolism by modulating nonpolar lipid biosynthesis through its interaction with Msn4p.

ATG15 encodes a phospholipase and is transcriptionally regulated by YAP1 in Saccharomyces cerevisiae.

Phospholipases play a vital role in maintaining membrane phospholipids. In this study, we found that deletion of the three major phospholipases B in Saccharomyces cerevisiae did not affect the hydrolysis of phospholipids, thus suggesting the presence of other, as yet unidentified, phospholipases. Indeed, in silico analysis of the S. cerevisiae genome identified 13 proteins that contain a conserved, putative serine hydrolase motif. In addition, expression profiling revealed that ATG15 (Autophagy 15) was highly expressed in the phospholipase B triple mutant. ATG15 encodes a phospholipase that preferentially hydrolyzes phosphatidylserine. Our analysis of the ATG15 promoter identified binding sites for Yap1p. In vivo and in vitro results showed that Yap1p positively regulates ATG15 expression. Collectively, we demonstrate that Atg15p is a phosphatidylserine lipase and that Yap1p activates the expression of ATG15 during autophagy.

Misregulation of a DDHD Domain-containing Lipase Causes Mitochondrial Dysfunction in Yeast.

The DDHD domain-containing proteins, which belong to the intracellular phospholipase A1 (iPLA1) family, have been predicted to be involved in phospholipid metabolism, lipid trafficking, membrane turnover, and signaling. Defective cardiolipin (CL), phosphatidylethanolamine, and phosphatidylglycerol remodeling cause Barth syndrome and mitochondrial dysfunction. Here, we report that Yor022c is a Ddl1 (DDHD domain-containing lipase 1) that hydrolyzes CL, phosphatidylethanolamine, and phosphatidylglycerol. Ddl1 has been implicated in the remodeling of mitochondrial phospholipids and CL degradation. Our data also suggested that the accumulation of monolysocardiolipin is deleterious to the cells. We show that Aft1 and Aft2 transcription factors antagonistically regulate the DDL1 gene. This study reveals that the misregulation of DDL1 by Aft1/2 transcription factors alters CL metabolism and causes mitochondrial dysfunction in the cells. In humans, mutations in the DDHD1 and DDHD2 genes cause specific types of hereditary spastic paraplegia (SPG28 and SPG54, respectively), and the yeast DDL1-defective strain produces similar phenotypes of hereditary spastic paraplegia (mitochondrial dysfunction and defects in lipid metabolism). Therefore, the DDL1-defective strain could be a good model system for understanding hereditary spastic paraplegia.