Teresa M. Dunn

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.

An Inositolphosphorylceramide Synthase Is Involved in Regulation of Plant Programmed Cell Death Associated with Defense in Arabidopsis

The Arabidopsis thaliana resistance gene RPW8 triggers the hypersensitive response (HR) to restrict powdery mildew infection via the salicylic acid–dependent signaling pathway. To further understand how RPW8 signaling is regulated, we have conducted a genetic screen to identify mutations enhancing RPW8-mediated HR-like cell death (designated erh). Here, we report the isolation and characterization of the Arabidopsis erh1 mutant, in which the At2g37940 locus is knocked out by a T-DNA insertion. Loss of function of ERH1 results in salicylic acid accumulation, enhanced transcription of RPW8 and RPW8-dependent spontaneous HR-like cell death in leaf tissues, and reduction in plant stature. Sequence analysis suggests that ERH1 may encode the long-sought Arabidopsis functional homolog of yeast and protozoan inositolphosphorylceramide synthase (IPCS), which converts ceramide to inositolphosphorylceramide. Indeed, ERH1 is able to rescue the yeast aur1 mutant, which lacks the IPCS, and the erh1 mutant plants display reduced (∼53% of wild type) levels of leaf IPCS activity, indicating that ERH1 encodes a plant IPCS. Consistent with its biochemical function, the erh1 mutation causes ceramide accumulation in plants expressing RPW8. These data reinforce the concept that sphingolipid metabolism (specifically, ceramide accumulation) plays an important role in modulating plant programmed cell death associated with defense.

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 50 mM Ca2+, but not 50 mM 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.

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

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 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.

The Essential Nature of Sphingolipids in Plants as Revealed by the Functional Identification and Characterization of the Arabidopsis LCB1 Subunit of Serine Palmitoyltransferase

Serine palmitoyltransferase (SPT) catalyzes the first step of sphingolipid biosynthesis. In yeast and mammalian cells, SPT is a heterodimer that consists of LCB1 and LCB2 subunits, which together form the active site of this enzyme. We show that the predicted gene for Arabidopsis thaliana LCB1 encodes a genuine subunit of SPT that rescues the sphingolipid long-chain base auxotrophy of Saccharomyces cerevisiae SPT mutants when coexpressed with Arabidopsis LCB2. In addition, homozygous T-DNA insertion mutants for At LCB1 were not recoverable, but viability was restored by complementation with the wild-type At LCB1 gene. Furthermore, partial RNA interference (RNAi) suppression of At LCB1 expression was accompanied by a marked reduction in plant size that resulted primarily from reduced cell expansion. Sphingolipid content on a weight basis was not changed significantly in the RNAi suppression plants, suggesting that plants compensate for the downregulation of sphingolipid synthesis by reduced growth. At LCB1 RNAi suppression plants also displayed altered leaf morphology and increases in relative amounts of saturated sphingolipid long-chain bases. These results demonstrate that plant SPT is a heteromeric enzyme and that sphingolipids are essential components of plant cells and contribute to growth and development.

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.

Metabolic response to iron deficiency in Saccharomyces cerevisiae.

Iron is an essential cofactor for enzymes involved in numerous cellular processes, yet little is known about the impact of iron deficiency on cellular metabolism or iron proteins. Previous studies have focused on changes in transcript and proteins levels in iron-deficient cells, yet these changes may not reflect changes in transport activity or flux through a metabolic pathway. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine which biosynthetic processes are altered when iron availability falls. Iron deficiency led to changes in glucose metabolism, amino acid biosynthesis, and lipid biosynthesis that were due to deficiencies in specific iron-dependent enzymes. Iron-sulfur proteins exhibited loss of iron cofactors, yet amino acid synthesis was maintained. Ergosterol and sphingolipid biosynthetic pathways had blocks at points where heme and diiron enzymes function, whereas Ole1, the essential fatty acid desaturase, was resistant to iron depletion. Iron-deficient cells exhibited depletion of most iron enzyme activities, but loss of activity during iron deficiency did not consistently disrupt metabolism. Amino acid homeostasis was robust, but iron deficiency impaired lipid synthesis, altering the properties and functions of cellular membranes.