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 cells require two genes, CSG1/SUR1 and CSG2, for growth in 50 mM Ca2+, but not 50 mM Sr2+. CSG2 was previously shown to be required for the mannosylation of inositolphosphorylceramide (IPC) to form mannosylinositolphosphorylceramide (MIPC). Here we demonstrate that SUR1/CSG1 is both genetically and biochemically related to CSG2. Like CSG2, SUR1/CSG1 is required for IPC mannosylation. A 93-amino acid stretch of Csg1p shows 29% identity with the alpha-1, 6-mannosyltransferase encoded by OCH1. The SUR1/CSG1 gene is a dose-dependent suppressor of the Ca(2+)-sensitive phenotype of the csg2 mutant, but overexpression of CSG2 does not suppress the Ca2+ sensitivity of the csg1 mutant. The csg1 and csg2 mutants display normal growth in YPD, indicating that mannosylation of sphingolipids is not essential. Increased osmolarity of the growth medium increases the Ca2+ tolerance of csg1 and csg2 mutant cells, suggesting that altered cell wall synthesis causes Ca(2+)-induced death. Hydroxylation of IPC-C to form IPC-D requires CCC2, a gene encoding an intracellular Cu2+ transporter. Increased expression of CCC2 or increased Cu2+ concentration in the growth medium enhances the Ca2+ tolerance of csg1 mutants, suggesting that accumulation of IPC-C renders csg1 cells Ca2+ sensitive.

It was recently demonstrated that mutations in the human SPTLC1 gene, encoding the Lcb1p subunit of serine palmitoyltransferase (SPT), cause hereditary sensory neuropathy type I (1, 2). As a member of the subfamily of pyridoxal 5-phosphate enzymes known as the ␣-oxoamine synthases, serine palmitoyltransferase catalyzes the committed step of sphingolipid synthesis. The residues that are mutated to cause hereditary sensory neuropathy type I reside in a highly conserved region of Lcb1p that is predicted to be a catalytic domain of Lcb1p on the basis of alignments with other members of the ␣-oxoamine synthase family. We found that the corresponding mutations in the LCB1 gene of Saccharomyces cerevisiae reduce serine palmitoyltransferase activity. These mutations are dominant and decrease serine palmitoyltransferase activity by 50% when the wild-type and mutant LCB1 alleles are coexpressed. We also show that serine palmitoyltransferase is an Lcb1p⅐Lcb2p heterodimer and that the mutated Lcb1p proteins retain their ability to interact with Lcb2p. Modeling studies suggest that serine palmitoyltransferase is likely to have a single active site that lies at the Lcb1p⅐Lcb2p interface and that the mutations in Lcb1p reside near the lysine in Lcb2p that is expected to form the Schiff's base with the pyridoxal 5-phosphate cofactor. Furthermore, mutations in this lysine and in a histidine residue that is also predicted to be important for pyridoxal 5-phosphate binding to Lcb2p also dominantly inactivate SPT similar to the hereditary sensory neuropathy type 1-like mutations in Lcb1p.

Saccharomyces cerevisiae mutants lacking Scs7p fail to accumulate the inositolphosphorylceramide (IPC) species, IPC‐C, which is the predominant form found in wild‐type cells. Instead scs7 mutants accumulate an IPC‐B species believed to be unhydroxylated on the amide‐linked C26‐fatty acid. Elimination of the SCS7 gene suppresses the Ca2+‐sensitive phenotype of csg1 and csg2 mutants. The CSG1 and CSG2 genes are required for mannosylation of IPC‐C and accumulation of IPC‐C by the csg mutants renders them Ca2+‐sensitive. The SCS7 gene encodes a protein that contains both a cytochrome b5‐like domain and a domain that resembles the family of cytochrome b5‐dependent enzymes that use iron and oxygen to catalyse desaturation or hydroxylation of fatty acids and sterols. Scs7p is therefore likely to be the enzyme that hydroxylates the C26‐fatty acid of IPC‐C. © 1998 John Wiley & Sons, Ltd.

Cashew nut seeds were subjected to processing including autoclaving (121 degrees C for 5, 10, 20, and 30 min), blanching (100 degrees C for 1, 4, 7, and 10 min), microwave heating (1 and 2 min each at 500 and 1000 W), dry roasting (140 degrees C for 20 and 30 min; 170 degrees C for 15 and 20 min; and 200 degrees C for 10 and 15 min), gamma-irradiation (1, 5, 10, and 25 kGy), and pH (1, 3, 5, 7, 9, 11, and 13). Proteins from unprocessed and processed cashew nut seeds were probed for stability using anti-Ana o 2 rabbit polyclonal antibodies and mouse monoclonal antibodies directed against Ana o 1, Ana o 2, and Ana o 3 as detection agents. Results indicate that Ana o 1, Ana o 2, and Ana o 3 are stable regardless of the processing method to which the nut seeds are subjected.