Ken Gable

Hydroxylation of Saccharomyces cerevisiae Ceramides Requires Sur2p and Scs7p

The Saccharomyces cerevisiae SCS7 andSUR2 genes are members of a gene family that encodes enzymes that desaturate or hydroxylate lipids. Sur2p is required for the hydroxylation of C-4 of the sphingoid moiety of ceramide, and Scs7p is required for the hydroxylation of the very long chain fatty acid. Neither SCS7 nor SUR2 are essential for growth, and lack of the Scs7p- or Sur2p-dependent hydroxylation does not prevent the synthesis of mannosyldiinositolphosphorylceramide, the mature sphingolipid found in yeast. Deletion of either gene suppresses the Ca2+-sensitive phenotype ofcsg2Δ mutants, which arises from overaccumulation of inositolphosphorylceramide due to a defect in sphingolipid mannosylation. Characterization of scs7 andsur2 mutants is expected to provide insight into the function of ceramide hydroxylation.

Tsc13p Is Required for Fatty Acid Elongation and Localizes to a Novel Structure at the Nuclear-Vacuolar Interface in Saccharomyces cerevisiae

ABSTRACT The TSC13/YDL015c gene was identified in a screen for suppressors of the calcium sensitivity of csg2Δ mutants that are defective in sphingolipid synthesis. The fatty acid moiety of sphingolipids in Saccharomyces cerevisiae is a very long chain fatty acid (VLCFA) that is synthesized by a microsomal enzyme system that lengthens the palmitate produced by cytosolic fatty acid synthase by two carbon units in each cycle of elongation. TheTSC13 gene encodes a protein required for elongation, possibly the enoyl reductase that catalyzes the last step in each cycle of elongation. The tsc13 mutant accumulates high levels of long-chain bases as well as ceramides that harbor fatty acids with chain lengths shorter than 26 carbons. These phenotypes are exacerbated by the deletion of either the ELO2 or ELO3gene, both of which have previously been shown to be required for VLCFA synthesis. Compromising the synthesis of malonyl coenzyme A (malonyl-CoA) by inactivating acetyl-CoA carboxylase in atsc13 mutant is lethal, further supporting a role of Tsc13p in VLCFA synthesis. Tsc13p coimmunoprecipitates with Elo2p and Elo3p, suggesting that the elongating proteins are organized in a complex. Tsc13p localizes to the endoplasmic reticulum and is highly enriched in a novel structure marking nuclear-vacuolar junctions.

The Saccharomyces cerevisiae TSC10/YBR265w gene encoding 3-ketosphinganine reductase is identified in a screen for temperature-sensitive suppressors of the Ca2+-sensitive csg2Delta mutant.

Saccharomyces cerevisiae csg2Δmutants accumulate the sphingolipid inositolphosphorylceramide, which renders the cells Ca2+-sensitive. Temperature-sensitive mutations that suppress the Ca2+ sensitivity ofcsg2Δ mutants were isolated and characterized to identify genes that encode sphingolipid synthesis enzymes. Thesetemperature-sensitive csg2Δ suppressors (tsc) fall into 15 complementation groups. TheTSC10/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 fromtsc10 mutants have low 3-ketosphinganine reductase activity. His6-tagged Tsc10p was expressed inEscherichia coli and isolated by nickel-nitrilotriacetic 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.

SUR1 (CSG1 / BCL21), a gene necessary for growth of Saccharomyces cerevisiae in the presence of high Ca2+ concentrations at 37° C, is required for mannosylation of inositolphosphorylceramide

Saccharomyces cerevisiae cells require two genes, CSG1/SUR1 and CSG2, for growth in 50u2009mM Ca2+, but not 50u2009mM Sr2+. CSG2 was previously shown to be required for the mannosylation of inositol-phosphorylceramide (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 α-1, 6-mannosyltransferase encoded by OCH1. The SUR1/CSG1 gene is a dose-dependent suppressor of the Ca2+-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 Ca2+-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.

The saccharomyces cerevisiae YBR159w gene encodes the 3-ketoreductase of the microsomal fatty acid elongase

The YBR159w gene encodes the major 3-ketoreductase activity of the elongase system of enzymes required for very long-chain fatty acid (VLCFA) synthesis. Mutants lacking the YBR159w gene display many of the phenotypes that have previously been described for mutants with defects in fatty acid elongation. These phenotypes include reduced VLCFA synthesis, accumulation of high levels of dihydrosphingosine and phytosphingosine, and accumulation of medium-chain ceramides. In vitroelongation assays confirm that the ybr159Δ mutant is deficient in the reduction of the 3-ketoacyl intermediates of fatty acid elongation. The ybr159Δ mutant also displays reduced dehydration of the 3-OH acyl intermediates of fatty acid elongation, suggesting that Ybr159p is required for the stability or function of the dehydratase activity of the elongase system. Green fluorescent protein-tagged Ybr159p co-localizes and co-immunoprecipitates with other elongating enzymes, Elo3p and Tsc13p. Whereas VLCFA synthesis is essential for viability, the ybr159Δ mutant cells are viable (albeit very slowly growing) and do synthesize some VLCFA. This suggested that a functional ortholog of Ybr159p exists that is responsible for the residual 3-ketoreductase activity. By disrupting the orthologs of Ybr159w in the ybr159Δmutant we found that the ybr159Δayr1Δ double mutant was inviable, suggesting that Ayr1p is responsible for the residual 3-ketoreductase activity.

Mutations in the yeast LCB1 and LCB2 genes, including those corresponding to the hereditary sensory neuropathy type I mutations, dominantly inactivate serine palmitoyltransferase

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 α-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 α-oxoamine synthase family. We found that the corresponding mutations in the LCB1 gene ofSaccharomyces cerevisiae reduce serine palmitoyltransferase activity. These mutations are dominant and decrease serine palmitoyltransferase activity by 50% when the wild-type and mutantLCB1 alleles are coexpressed. We also show that serine palmitoyltransferase is an Lcb1p·Lcb2p 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 Lcb1p·Lcb2p interface and that the mutations in Lcb1p reside near the lysine in Lcb2p that is expected to form the Schiffs 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.

A Saccharomyces cerevisiae Gene Required for Heterologous Fatty Acid Elongase Activity Encodes a Microsomal β-Keto-reductase

A number of Saccharomyces cerevisiaemembrane-bound oxidoreductases were examined for potential roles in microsomal fatty acid elongation, by assaying heterologous elongating activities in individual deletion mutants. One yeast gene, YBR159w, was identified as being required for activity of both theCaenorhabditis elegans elongase PEA1 (F56H11.4) and theArabidopsis thaliana elongase FAE1. Ybr159p shows some limited homology to human steroid dehydrogenases and is a member of the short-chain alcohol dehydrogenase superfamily. Disruption of YBR159w is not lethal, in contrast to previous reports, although the mutants are slow growing and display high temperature sensitivity. Both Ybr159p and an Arabidopsis homologue were shown to restore heterologous elongase activities when expressed in ybr159Δ mutants. Biochemical characterization of microsomal preparations fromybr159Δ cells revealed a primary perturbation in β-ketoacyl reduction, confirming the assignment of YBR159w as encoding a component of the microsomal elongase.

The Topology of the Lcb1p Subunit of Yeast Serine Palmitoyltransferase

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 ∼60 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 α-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.

Overexpression of the Wild-Type SPT1 Subunit Lowers Desoxysphingolipid Levels and Rescues the Phenotype of HSAN1

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 (tgSPTLC1C133W) 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 tgSPTLC1C133W, but not in the wild-type, double-transgenic tgSPTLC1WT + C133W or SPTLC1+/− 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.

Mutations in the Lcb2p subunit of serine palmitoyltransferase eliminate the requirement for the TSC3 gene in Saccharomyces cerevisiae.

Serine palmitoyltransferase catalyses the committed step in sphingolipid synthesis, the condensation of serine with palmitoyl‐CoA to form 3‐ketosphinganine. Two proteins, Lcb1p and Lcb2p, are essential for enzyme activity and a third protein, the 80‐amino acid Tsc3p, stimulates the activity of serine palmitoyltransferase several‐fold. Tsc3p physically associates with a complex of Lcb1p–Lcb2p and stimulates enzyme activity post‐translationally, but its precise function is not known. Tsc3p is essential for cell viability only at elevated temperatures, although serine palmitoyltransferase activity is reduced in the tsc3Δ mutant, even at permissive growth temperatures. Tsc3p is apparently not required for any essential process besides stimulation of serine palmitoyltransferase at 37°C, since providing sphingoid bases to the growth medium reverses the temperature‐sensitive growth phenotype of the tsc3Δ mutant. To gain further insight into the function of Tsc3p, suppressor mutants that eliminate the Tsc3p requirement for growth at 37°C were isolated and characterized. These studies show that dominant mutations in the Lcb2p subunit of serine palmitoyltransferase suppress the temperature‐sensitive growth phenotype of the tsc3Δ null mutant by increasing the Tsc3p‐independent serine palmitoyltransferase activity. Copyright