The Saccharomyces cerevisiae TSC10/YBR265w Gene Encoding 3-Ketosphinganine Reductase Is Identified in a Screen for Temperature-sensitive Suppressors of the Ca2+-sensitive csg2Δ Mutant

Abstract

Saccharomyces cerevisiae csg2⌬ mutants accumulate the sphingolipid inositolphosphorylceramide, which renders the cells Ca 2؉ -sensitive. Temperature-sensitive mutations that suppress the Ca 2؉ sensitivity of csg2⌬ mutants were isolated and characterized to identify genes that encode sphingolipid synthesis enzymes. These temperature-sensitive csg2⌬ suppressors (tsc) fall into 15 complementation groups. The TSC10/YBR265w gene was found to encode 3-ketosphinganine reductase, the enzyme that catalyzes the second step in the synthesis of phytosphingosine, the long chain base found in yeast sphingolipids. 3-Ketosphinganine reductase (Tsc10p) is essential for growth in the absence of exogenous dihydrosphingosine or phytosphingosine. Tsc10p is a member of the short chain dehydrogenase/reductase protein family. The tsc10 mutants accumulate 3-ketosphinganine and microsomal membranes isolated from tsc10 mutants have low 3-ketosphinganine reductase activity. His 6 -tagged Tsc10p was expressed in Escherichia coli and isolated by nickelnitrilotriacetic acid column chromatography. The recombinant protein catalyzes the NADPH-dependent reduction of 3-ketosphinganine. These data indicate that Tsc10p is necessary and sufficient for catalyzing the NADPH-dependent reduction of 3-ketosphinganine to dihydrosphingosine.Sphingolipids are essential components of eukaryotic membranes. They are composed of a polar head attached to a hydrophobic ceramide base. Ceramide contains a fatty acid attached in amide linkage to a long chain base (LCB).

Alteration of complex sphingolipid composition and its physiological significance in yeast Saccharomyces cerevisiae lacking vacuolar ATPase

“…These results suggested that the loss of MIPC synthesis and the change in the hydroxylation levels in sphingolipids can broadly affect the phenotypes caused by the deletion of V-ATPase. As reported previously, sur1D csh1D cells exhibited supersensitivity to Ca 2+ , and the sensitivity was rescued by the deletion of SUR2 or SCS7 (Beeler et al, 1998;Zhao et al, 1994) (Fig. 7a, b).…”

“…S1). The Ca 2+ supersensitivity of MIPC synthesis-deficient mutants is caused by the accumulation of IPC-C (Beeler et al, 1998;Zhao et al, 1994), and the cell death under nitrogen starvation due to the loss of MIPC synthesis is rescued by the deletion of SUR2 or myriocin, both of which prevent the accumulation of IPC-C (Yamagata et al, 2013). However, the growth defect of sur1D csh1D vma2D cells at pH 7.2 was not suppressed on the deletion of SUR2 or SCS7 (Fig.…”

“…It was previously reported that some abnormal phenotypes found in MIPC synthesis-deficient mutants are caused by accumulation of IPC-C, but not by loss of MIPC itself. For example, the Ca 2+ -sensitive phenotype of csg2D cells is suppressed by mutation of SUR2, SCS7, LCB1, LCB2, TSC3 or TSC10, all of which suppress the accumulation of IPC-C (Beeler et al, 1998;Zhao et al, 1994). Thus, we investigated whether or not the growth defect of sur1D csh1D vma2D cells at pH 7.2 is suppressed by the deletion of SUR2 or SCS7, either of which causes loss of IPC-C (Haak et al, 1997;Uemura et al, 2014).…”

Csg1p and Newly Identified Csh1p Function in Mannosylinositol Phosphorylceramide Synthesis by Interacting with Csg2p

“…Other suppressors of Ca 2ϩ sensitivity in the csg2 mutant were found to be genes involved in signal transduction. These included genes encoding the protein kinase TOR2 and the phosphatidylinositol-4-phosphate 5-kinase MSS4 (9). Tor2p functions in a nutrient-sensing signaling cascade and in cell cycle-dependent reorganization of actin cytoskeleton (26 -30).…”

“…The Ca 2ϩ -sensitive phenotype of the csg2 mutant was suppressed by several mutations that caused a reduction in the IPC-C concentrations (6). These mutations included genes involved in long chain base synthesis (lcb1, lcb2, tsc3, and tsc10) (6,9,15), palmitoyl-CoA synthesis (fas2) (9), elongation of very long chain fatty acids (tsc13) (9,16), conversion of DHS to phytosphingosine (sur2) (1), and hydroxylation of very long chain fatty acids (scs7) (1). Moreover, overproduction of Ccc2p, which may stimulate the conversion of IPC-C to IPC-D, also rescued the Ca 2ϩ -sensitive phenotype of the csg2 mutant (3).…”