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.

Global control for the synthesis of lipids constituting a bilayer of cell membranes is known to be with a small number of transcription factors called master transcriptional regulators, which target a wide range of genes encoding lipid metabolism enzymes and/or their regulators. Although master transcriptional regulators of glycerophospholipids and sterols have been identified in both yeast and mammals, this aspect of sphingolipid metabolism is not yet understood. In the present study, we identified the C2H2-type zinc finger transcription factor, Com2, as a master transcriptional regulator of sphingolipid metabolism in the budding yeast, Saccharomyces cerevisiae. The target of rapamycin complex 2 (TORC2)-activated protein kinase Ypk1 is known to regulate sphingolipid metabolism. Activated Ypk1 stimulates the activity of serine palmitoyl transferase (SPT), the first-step enzyme in sphingolipid biosynthesis, by phosphorylating and inhibiting Orm1/2, a negative regulator of SPT. This regulation of SPT activity is thought to be a major pathway in the regulation of sphingolipid metabolism. In the present study, we found that inhibition of sphingolipid synthesis upregulates the expression of Com2, which in turn leads to the concomitant expression of Ypk1. The upregulation of Ypk1 expression was found to be dependent on a putative Com2-binding site in the YPK1 promoter. Our results also suggested that Com2 senses intracellular sphingolipid levels through a pathway independent of TORC2-Ypk1-mediated sensing of sphingolipids. Our results revealed an additional layer of mechanistic regulation that allows cells to maintain appropriate levels of sphingolipid biosynthesis and to rapidly induce this process in response to environmental stresses.Significance StatementOne of the major regulatory mechanisms involved in the control of lipid metabolism in bilayers of biological membranes is regulation at the transcriptional level by master transcriptional regulators that control the transcription of genes encoding lipid metabolism enzymes and/or their regulators. In the present study, we identified the C2H2-type zinc finger transcription factor Com2 as a master transcriptional regulator in sphingolipid metabolism. We found that Com2 regulates sphingolipid metabolism by transcriptionally controlling the expression of Ypk1, which regulates Orm1/2, a negative regulator of serine palmitoyl transferase, the first-step enzyme in sphingolipid biosynthesis, through phosphorylation. Our study revealed a new layer of regulation that allows the maintenance of an appropriate level of sphingolipid biosynthesis for a rapid response to environmental stresses.

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