Ram Rajasekharan

Responses to phosphate deprivation in yeast cells

Inorganic phosphate is an essential nutrient because it is required for the biosynthesis of nucleotides, phospholipids and metabolites in energy metabolism. During phosphate starvation, phosphatases play a major role in phosphate acquisition by hydrolyzing phosphorylated macromolecules. In Saccharomyces cerevisiae, PHM8 (YER037W), a lysophosphatidic acid phosphatase, plays an important role in phosphate acquisition by hydrolyzing lysophosphatidic acid and nucleotide monophosphate that results in accumulation of triacylglycerol and nucleotides under phosphate limiting conditions. Under phosphate limiting conditions, it is transcriptionally regulated by Pho4p, a phosphate-responsive transcription factor. In this review, we focus on triacylglycerol metabolism in transcription factors deletion mutants involved in phosphate metabolism and propose a link between phosphate and triacylglycerol metabolism. Deletion of these transcription factors results in an increase in triacylglycerol level. Based on these observations, we suggest that PHM8 is responsible for the increase in triacylglycerol in phosphate metabolising gene deletion mutants.

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.

Exploring Triacylglycerol Biosynthetic Pathway in Developing Seeds of Chia ( Salvia hispanica L.): A Transcriptomic Approach

Chia (Salvia hispanica L.), a member of the mint family (Lamiaceae), is a rediscovered crop with great importance in health and nutrition and is also the highest known terrestrial plant source of heart-healthy omega-3 fatty acid, alpha linolenic acid (ALA). At present, there is no public genomic information or database available for this crop, hindering research on its genetic improvement through genomics-assisted breeding programs. The first comprehensive analysis of the global transcriptome profile of developing Salvia hispanica L. seeds, with special reference to lipid biosynthesis is presented in this study. RNA from five different stages of seed development was extracted and sequenced separately using the Illumina GAIIx platform. De novo assembly of processed reads in the pooled transcriptome using Trinity yielded 76,014 transcripts. The total transcript length was 66,944,462 bases (66.9 Mb), with an average length of approximately 880 bases. In the molecular functions category of Gene Ontology (GO) terms, ATP binding and nucleotide binding were found to be the most abundant and in the biological processes category, the metabolic process and the regulation of transcription-DNA-dependent and oxidation-reduction process were abundant. From the EuKaryotic Orthologous Groups of proteins (KOG) classification, the major category was “Metabolism” (31.97%), of which the most prominent class was ‘carbohydrate metabolism and transport’ (5.81% of total KOG classifications) followed by ‘secondary metabolite biosynthesis transport and catabolism’ (5.34%) and ‘lipid metabolism’ (4.57%). A majority of the candidate genes involved in lipid biosynthesis and oil accumulation were identified. Furthermore, 5596 simple sequence repeats (SSRs) were identified. The transcriptome data was further validated through confirmative PCR and qRT-PCR for select lipid genes. Our study provides insight into the complex transcriptome and will contribute to further genome-wide research and understanding of chia. The identified novel UniGenes will facilitate gene discovery and creation of genomic resource for this crop.

ZAP1-mediated modulation of triacylglycerol levels in yeast by transcriptional control of mitochondrial fatty acid biosynthesis.

The transcriptional activator Zap1p maintains zinc homeostasis in Saccharomyces cerevisiae. In this study, we examined the role of Zap1p in triacylglycerol (TAG) metabolism. The expression of ETR1 is reduced in zap1Δ. The altered expression of ETR1 results in reduced mitochondrial fatty acid biosynthesis and reduction in lipoic acid content in zap1Δ. The transcription factor Zap1 positively regulates ETR1 expression. Deletion of ETR1 also causes the accumulation of TAG, and the introduction of ETR1 in zap1Δ strain rescues the TAG level. These results demonstrated that the compromised mitochondrial fatty acid biosynthesis causes a reduction in lipoic acid and loss of mitochondrial function in zap1Δ. Functional mitochondria are required for the ATP production and defect in mitochondria slow down the process which may channeled carbon towards lipid biosynthesis and stored in the form of TAG.

PHO4 transcription factor regulates triacylglycerol metabolism under low-phosphate conditions in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, PHM8 encodes a phosphatase that catalyses the dephosphorylation of lysophosphatidic acids to monoacylglycerol and nucleotide monophosphate to nucleoside and releases free phosphate. In this report, we investigated the role of PHM8 in triacylglycerol metabolism and its transcriptional regulation by a phosphate responsive transcription factor Pho4p under low‐phosphate conditions. We found that the wild‐type (BY4741) cells accumulate triacylglycerol and the expression of PHM8 was high under low‐phosphate conditions. Overexpression of PHM8 in the wild‐type, phm8Δ and quadruple phosphatase mutant (pah1Δdpp1Δlpp1Δapp1Δ) caused an increase in the triacylglycerol levels. However, the introduction of the PHM8 deletion into the quadruple phosphatase mutant resulted in a reduction in triacylglycerol levels and LPA phosphatase activity. The transcriptional activator Pho4p binds to the PHM8 promoter under low‐phosphate conditions, activating PHM8 expression, which leads to the formation of monoacylglycerol from LPA. The synthesized monoacylglycerol is acylated to diacylglycerol by Dga1p, which is further acylated to triacylglycerol by the same enzyme.

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.

The transcription factor GCN4 regulates PHM8 and alters triacylglycerol metabolism in Saccharomyces cerevisiae

PHM8 is a very important enzyme in nonpolar lipid metabolism because of its role in triacylglycerol (TAG) biosynthesis under phosphate stress conditions. It is positively regulated by the PHO4 transcription factor under low phosphate conditions; however, its regulation has not been explored under normal physiological conditions. General control nonderepressible (GCN4), a basic leucine-zipper transcription factor activates the transcription of amino acids, purine biosynthesis genes and many stress response genes under various stress conditions. In this study, we demonstrate that the level of TAG is regulated by the transcription factor GCN4. GCN4 directly binds to its consensus recognition sequence (TGACTC) in the PHM8 promoter and controls its expression. The analysis of cells expressing the PPHM8-lacZ reporter gene showed that mutations (TGACTC–GGGCCC) in the GCN4-binding sequence caused a significant increase in β-galactosidase activity. Mutation in the GCN4 binding sequence causes an increase in PHM8 expression, lysophosphatidic acid phosphatase activity and TAG level. PHM8, in conjunction with DGA1, a mono- and diacylglycerol transferase, controls the level of TAG. These results revealed that GCN4 negatively regulates PHM8 and that deletion of GCN4 causes de-repression of PHM8, which is responsible for the increased TAG content in gcn4∆ cells.

The m 6 A 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.

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.

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.