Serine palmitoyltransferase (SPT) catalyzes the first committed step in sphingolipid biosynthesis. In yeast, SPT is composed of a heterodimer of 2 highly-related subunits, Lcb1p and Lcb2p, and a third subunit, Tsc3p, which increases enzyme activity markedly and is required for growth at elevated temperatures. Higher eukaryotic orthologs of Lcb1p and Lcb2p have been identified, but SPT activity is not highly correlated with coexpression of these subunits and no ortholog of Tsc3p has been identified. Here, we report the discovery of 2 proteins, ssSPTa and ssSPTb, which despite sharing no homology with Tsc3p, each substantially enhance the activity of mammalian SPT expressed in either yeast or mammalian cells and therefore define an evolutionarily conserved family of low molecular weight proteins that confer full enzyme activity. The 2 ssSPT isoforms share a conserved hydrophobic central domain predicted to reside in the membrane, and each interacts with both hLCB1 and hLCB2 as assessed by positive split ubiquitin 2-hybrid analysis. The presence of these small subunits, along with 2 hLCB2 isofoms, suggests that there are 4 distinct human SPT isozymes. When each SPT isozyme was expressed in either yeast or CHO LyB cells lacking endogenous SPT activity, characterization of their in vitro enzymatic activities, and long-chain base (LCB) profiling revealed differences in acyl-CoA preference that offer a potential explanation for the observed diversity of LCB seen in mammalian cells.

The autosomal dominant peripheral sensory neuropathy HSAN1 results from mutations in the LCB1 subunit of serine palmitoyltransferase (SPT). Serum from patients and transgenic mice expressing a disease-causing mutation (C133W) contain elevated levels of 1-deoxysphinganine (1-deoxySa), which presumably arise from inappropriate condensation of alanine with palmitoyl-CoA. Mutant heterodimeric SPT is catalytically inactive. However, mutant heterotrimeric SPT has ϳ10 -20% of wild-type activity and supports growth of yeast cells lacking endogenous SPT. In addition, long chain base profiling revealed the synthesis of significantly more 1-deoxySa in yeast and mammalian cells expressing the heterotrimeric mutant enzyme than in cells expressing wild-type enzyme. Wild-type and mutant enzymes had similar affinities for serine. Surprisingly, the enzymes also had similar affinities for alanine, indicating that the major affect of the C133W mutation is to enhance activation of alanine for condensation with the acyl-CoA substrate. In vivo synthesis of 1-deoxySa by the mutant enzyme was proportional to the ratio of alanine to serine in the growth media, suggesting that this ratio can be used to modulate the relative synthesis of sphinganine and 1-deoxySa. By expressing SPT as a single-chain fusion protein to ensure stoichiometric expression of all three subunits, we showed that GADD153, a marker for endoplasmic reticulum stress, was significantly elevated in cells expressing mutant heterotrimers. GADD153 was also elevated in cells treated with 1-deoxySa. Taken together, these data indicate that the HSAN1 mutations perturb the active site of SPT resulting in a gain of function that is responsible for the HSAN1 phenotype. Serine palmitoyltransferase (SPT)3 catalyzes the committed and rate-limiting step in sphingolipid biosynthesis. Until recently, it was believed that there was a single isozyme of this enzyme containing two subunits, LCB1 and LCB2, with the catalytic domain located at the interface and composed of residues from each subunit. However, Hornemann et al. showed that there was a second isoform of LCB2 (1). More recently, we identified two highly related isoforms of a third subunit, ssSPTa and ssSPTb, which are essential for maximal enzymatic activity (2). Thus, there are at least four potential SPT isozymes, each containing a common hLCB1 subunit and one of each of the two hLCB2 and ssSPT subunits. Examination of the acyl-CoA preferences of the hLCB1/LCB2a and hLCB1/LCB2b heterodimers and the hLCB1/LCB2/ssSPT heterotrimers showed that both the LCB2 isoform and the ssSPT isoform conferred different acyl-CoA preferences (2, 3), revealing a previously unappreciated complexity in SPT substrate selectivity, and the corresponding long chain base (LCB) products.Mutations in the hLCB1 subunit of SPT have been demonstrated to cause the autosomal dominant late-onset hereditary sensory neuropathy, type I (HSAN1) (4 -6). In addition, an HSAN1-like phenotype is seen in transgenic mice overexpressing the LCB1 C133W mutation (7). Beca...

Growth of a human leukemic T-cell line (CEM C7) in 10(-6) M dexamethasone results in inhibition of growth and rapid loss of cell viability after a delay of approximately 18 to 24 hours. Analysis of dexamethasone-treated cells by flow-microfluorometry showed that they were arrested in the G1 phase of the cell cycle. Loss of cell viability began at the same time as G1 accumulation was first detectable, and 20% of all cells were found to be blocked in G1 at this time suggesting that loss of viability and G1 arrest were coincident events. Half-maximal and maximal effects on both viability and G1 arrest after 48 hours in steroid were nearly identical with respect to steroid concentration and corresponded to half-maximal and full occupancy of glucocorticoid specific receptor by hormone, consistent with a glucocorticoid receptor mediated mechanism for both phenomena. Most non-viable cells were arrested in G1, and accumulation of cells in G1 was irreversible; removal of steroid in the presence of colcemid did not result in a decreased fraction of G1 cells. Furthermore, dexamethasone treatment did not protect cells against the effects of 33258 Hoechst-amplified killing of bromodeoxyuridine substituted cells exposed to light. These results show that dexamethasone arrests these leukemic cells in G1 and strongly suggest that dexamethasone-treated cells are killed upon entry into G1.

Mutations in the SPTLC1 subunit of serine palmitoyltransferase (SPT) cause an adult-onset, hereditary sensory, and autonomic neuropathy type I (HSAN1). We previously reported that mice bearing a transgene-expressing mutant SPTLC1 (tgSPTLC1 C133W ) show a reduction in SPT activity and hyperpathia at 10 months of age. Now analyzed at a later age, we find these mice develop sensory loss with a distal small fiber neuropathy and peripheral myelinopathy. This phenotype is largely reversed when these mice are crossed with transgenic mice overexpressing wild-type SPTLC1 showing that the mutant SPTLC1 protein is not inherently toxic. Simple loss of SPT activity also cannot account for the HSAN1 phenotype, since heterozygous SPTLC1 knock-out mice have reduced SPT activity but are otherwise normal. Rather, the presence of two newly identified, potentially deleterious deoxysphingoid bases in the tgSPTLC1 C133W , but not in the wild-type, double-transgenic tgSPTLC1 WT ϩ C133W or SPTLC1 ϩ/Ϫ mice, suggests that the HSAN1 mutations alter amino acid selectivity of the SPT enzyme such that palmitate is condensed with alanine and glycine, in addition to serine. This observation is consistent with the hypothesis that HSAN1 is the result of a gain-of-function mutation in SPTLC1 that leads to accumulation of a toxic metabolite.

The structural organization and topology of the Lcb1p subunit of yeast and mammalian serine palmitoyltransferases (SPT) were investigated. In the yeast protein, three membrane-spanning domains were identified by insertion of glycosylation and factor Xa cleavage sites at various positions. The first domain of the yeast protein, located between residues 50 and 84, was not required for the stability, membrane association, interaction with Lcb2p, or enzymatic activity. Deletion of the comparable domain of the mammalian protein SPTLC1 also had little effect on its function, demonstrating that this region is not required for membrane localization or heterodimerization with SPTLC2. The second and third membrane-spanning domains of yeast Lcb1p, located between residues 342 and 371 and residues 425 and 457, respectively, create a luminal loop of ϳ60 residues. In contrast to the first membrane-spanning domain, the second and third membrane-spanning domains were both required for Lcb1p stability. In addition, mutations in the luminal loop destabilized the SPT heterodimer indicating that this region of the protein is important for SPT structure and function. Mutations in the extreme carboxyl-terminal region of Lcb1p also disrupted heterodimer formation. Taken together, these data suggest that in contrast to other members of the ␣-oxoamine synthases that are soluble homodimers, the Lcb1p and Lcb2p subunits of the SPT heterodimer may interact in the cytosol, as well as within the membrane and/or the lumen of the endoplasmic reticulum.