Serine palmitoyltransferase catalyzes the first step of sphingolipid synthesis, condensation of serine and palmitoyl CoA to form the long chain base 3-ketosphinganine. The LCB1/TSC2 and LCB2/TSC1 genes encode homologous proteins of the ␣-oxoamine synthase family required for serine palmitoyltransferase activity. The other ␣-oxoamine synthases are soluble homodimers, but serine palmitoyltransferase is a membrane-associated enzyme composed of at least two subunits, Lcb1p and Lcb2p. Here, we report the characterization of a third gene, TSC3, required for optimal 3-ketosphinganine synthesis in Saccharomyces cerevisiae. S. cerevisiae cells lacking the TSC3 gene have a temperature-sensitive lethal phenotype that is reversed by supplying 3-ketosphinganine, dihydrosphingosine, or phytosphingosine in the growth medium. (SPT) 1 catalyzes the formation of 3-ketosphinganine from serine and palmitoyl CoA. This is the first committed step in the synthesis of ceramides and sphingolipids. This step is also believed to be ratelimiting, making it likely that regulation of SPT controls the rate of sphingolipid synthesis. For example, treatment of mammalian cells with sphingosine results in down-regulation of SPT activity (1). However, little is known about how the enzyme is regulated.Two genes, LCB1/TSC2 and LCB2/TSC1, are required for SPT activity (2-4). Both genes encode proteins that belong to a small subfamily of pyridoxal 5Ј-phosphate-dependent enzymes that catalyze the condensation of an amino acid and a carboxylic acid CoA thioester with concomitant decarboxylation of the amino acid. This ␣-oxoamine synthase subfamily includes 8-amino-7-oxononanoate synthase (AONS), 5-aminolevulinate synthase, 2-amino-oxobutyrate CoA ligase, and SPT. Although these enzymes share low overall sequence identity, the recently reported crystal structure of AONS reveals that several functionally important residues are highly conserved (5). The residues that are involved in pyridoxal phosphate binding, including the lysine that forms a Schiff's base with pyridoxal phosphate, are conserved in Lcb2p but not in Lcb1p. Therefore, although the AONS enzyme is a symmetrical homodimer with the active site at the subunit interface, Lcb1p and Lcb2p may form a heterodimer because both proteins are required for SPT activity. The yeast and mammalian Lcb2ps are more similar to each other than they are to the yeast and human Lcb1ps (2, 6, 7). Likewise, the yeast and mammalian Lcb1ps are more similar to each other than they are to their Lcb2p counterparts (7,8). SPT also differs from the other members of this enzyme family because it is membrane-associated. SPT appears to be located on the cytoplasmic side of the endoplasmic reticulum (9). In comparison to AONS, Lcb1p and Lcb2p each have amino-terminal extensions that contain potential membrane-spanning segments. However, it is not known whether these hydrophobic segments are important for membrane association.Attempts to increase SPT activity by over-expression of LCB1 and LCB2 have met with limited success (...
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).
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