Tsc3p Is an 80-Amino Acid Protein Associated with Serine Palmitoyltransferase and Required for Optimal Enzyme Activity

Abstract

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 (...

Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis

“…It has been suggested that LCB2 acts as the enzymatically active center, and LCB1 appears to stabilize the heterodimer [14]. The lcb1 mutations caused a substantially reduced level of the LCB2 protein in both a Chinese hamster ovary cell mutant line [43] and yeast cells [44], thus leading to lethality in both cases. Similarly, the lcb1-1 mutation causes an embryolethal phenotype, indicating an essential role of FBR11 in plant growth and development [41].…”

Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities

“…For determination of SPT activity, a reaction containing 50 mM Hepes (pH 8.1), 50 M pyridoxyl phosphate, 2 mM NADPH, 2 mM NADH, 10 mM L-[G-3 H]serine (10 Ci/mL), 75 or 100 M acyl-CoA, and 0.2-0.4 mg of microsomal membranes was incubated at 37°C for 10 min. The reaction was stopped and the products were extracted and quantified as described (4).…”

“…long-chain bases ͉ sphingolipids ͉ HSAN1 ͉ ssSPT O riginally thought to be composed of a single subunit, genetic analysis in yeast showed that eukaryotic serine palmitoyltransferase (SPT) is a heterodimer of 2 highly-related subunits, Lcb1p and Lcb2p (1)(2)(3), associated with a third small subunit, Tsc3p, that is required for maximal enzyme activity (4). Based on the crystal structure of 8-amino-7-oxononanoate synthase (5), and the identification of key catalytic residues in other ␣-oxoamine synthases, we proposed that SPT contains a single cytoplasmic catalytic site at the interface of a head-to-tail heterodimer of Lcb1p and Lcb2p comprised of residues from both subunits (6).…”

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

“…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).…”