Analysis of phosphorylated sphingolipid long-chain bases reveals potential roles in heat stress and growth control in Saccharomyces.

Sphingolipid long-chain bases and their phosphorylated derivatives, for example, sphingosine-1-phosphate in mammals, have been implicated as signaling molecules. The possibility that Saccharomyces cerevisiae cells also use long-chain-base phosphates to regulate cellular processes has only recently begun to be examined. Here we present a simple and sensitive procedure for analyzing and quantifying long-chain-base phosphates in S. cerevisiae cells. Our data show for the first time that phytosphingosine-1-phosphate (PHS-1-P) is present at a low but detectable level in cells grown on a fermentable carbon source at 25 degreesC, while dihydrosphingosine-1-phosphate (DHS-1-P) is only barely detectable. Shifting cells to 37 degreesC causes transient eight- and fivefold increases in levels of PHS-1-P and DHS-1-P, respectively, which peak after about 10 min. The amounts of both compounds return to the unstressed levels by 20 min after the temperature shift. These data are consistent with PHS-1-P and DHS-1-P being signaling molecules. Cells unable to break down long-chain-base phosphates, due to deletion of DPL1 and LCB3, show a 500-fold increase in PHS-1-P and DHS-1-P levels, grow slowly, and survive a 44 degreesC heat stress 10-fold better than parental cells. These and other data for dpl1 or lcb3 single-mutant strains suggest that DHS-1-P and/or PHS-1-P act as signals for resistance to heat stress. Our procedure should expedite experiments to determine how the synthesis and breakdown of these compounds is regulated and how the compounds mediate resistance to elevated temperature.

UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes.

The enzyme serine palmitoyltransferase (SPT; EC 2.3.1.50), which catalyzes the first committed and rate-limiting step in sphingolipid synthesis, is up-regulated in the epidermis as part of the homeostatic repair in response to permeability barrier perturbation. Moreover, UVB exposure, which also perturbs the barrier, up-regulates sphingolipid synthesis, but the basis for this increase is not known. The recent isolation of cDNAs for SPT (i.e., LCB1 and LCB2) allow molecular regulation studies to be performed. Therefore, we determined whether UVB exposure alters mRNA, protein, or activity levels for SPT in cultured human keratinocytes (CHKs) as a mechanism for regulation of epidermal sphingolipid synthesis. In CHK, transcripts for both LCB1 (3.0 kb) and LCB2 (2.3 kb) are evident by Northern blot analysis, and UVB exposure (23 mJ/cm2) induces a delayed 1.8 to 3.3-fold increase in LCB2 mRNA levels (P < 0.01) 48 h after treatment versus non-irradiated control cells. In contrast, neither LCB1 nor a second LCB2 transcript (8.0 kb) changed significantly. Likewise, Lcb2 protein levels (by Western blot analysis), as well as SPT activity, increase in parallel with the increased LCB2 mRNA. Finally, incorporation of [14C]-acetate into sphingolipids was increased significantly 48 h after UVB treatment. Together, these results demonstrate that CHKs respond to UVB by increasing sphingolipid synthesis, primarily through increases in both LCB2 mRNA and protein levels, leading to increased SPT activity. These results demonstrate one mechanism (UVB) whereby SPT is regulated at the molecular level, and suggest further that epidermis up-regulates sphingolipid synthesis at both the mRNA and protein levels in response to UVB.

Molecular cloning and functional characterization of murine sphingosine kinase.

Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.

The LCB4 (YOR171c) and LCB5 (YLR260w) genes of Saccharomyces encode sphingoid long chain base kinases.

Sphingolipid long chain bases (LCBs) and phosphorylated derivatives, particularly sphingosine 1-phosphate, are putative signaling molecules. To help elucidate the physiological roles of LCB phosphates, we identified two Saccharomyces cerevisiae genes, LCB4 (YOR171c) and LCB5 (YLR260w), which encode LCB kinase activity. This conclusion is based upon the synthesis of LCB kinase activity in Escherichia coli expressing either LCB gene. LCB4 encodes most (97%) Saccharomyces LCB kinase activity, with the remainder requiring LCB5. Log phase lcb4-deleted yeast cells make no LCB phosphates, showing that the Lcb4 kinase synthesizes all detectable LCB phosphates under these growth conditions. The Lcb4 and Lcb5 proteins are paralogs with 53% amino acid identity but are not related to any known protein, thus revealing a new class of lipid kinase. Two-thirds of the Lcb4 and one-third of the Lcb5 kinase activity are in the membrane fraction of yeast cells, a puzzling finding in that neither protein contains a membrane-localization signal. Both enzymes can use phytosphingosine, dihydrosphingosine, or sphingosine as substrate. LCB4 and LCB5 should be useful for probing the functions of LCB phosphates in S. cerevisiae. Potential mammalian cDNA homologs of the LCB kinase genes may prove useful in helping to understand the function of sphingosine 1-phosphate in mammals.

Inhibition of amino acid transport by sphingoid long chain bases in Saccharomyces cerevisiae.

Sphingoid long chain bases have many effects on cells including inhibition or stimulation of growth. The physiological significance of these effects is unknown in most cases. To begin to understand how these compounds inhibit growth, we have studied Saccharomyces cerevisiae cells. Growth of tryptophan (Trp-) auxotrophs was more strongly inhibited by phytosphingosine (PHS) than was growth of Trp+ strains, suggesting that PHS diminishes tryptophan uptake and starves cells for this amino acid. This hypothesis is supported by data showing that growth inhibition is relieved by increasing concentrations of tryptophan in the culture medium and by multiple copies of the TAT2 gene, encoding a high affinity tryptophan transporter. Measurement of tryptophan uptake shows that it is inhibited by PHS. Finally, PHS treatment induces the general control response, indicating starvation for amino acids. Multiple copies of TAT2 do not protect cells against two other cationic lipids, stearylamine, and sphingosine, indicating that the effect of PHS on tryptophan utilization is specific. Other data demonstrate that PHS reduces uptake of leucine, histidine, and proline by specific transporters. Our data suggest that PHS targets proteins in the amino acid transporter family but not other distantly related membrane transporters, including those necessary for uptake of adenine and uracil.

Synthesis of mannose-(inositol-P)2-ceramide, the major sphingolipid in Saccharomyces cerevisiae, requires the IPT1 (YDR072c) gene.

Knowledge of the Saccharomyces cerevisiae genes and proteins necessary for sphingolipid biosynthesis is far from complete. Such information should expedite studies of pathway regulation and sphingolipid functions. Using the Aur1 protein sequence, recently identified as necessary for synthesis of the sphingolipid inositol-P-ceramide (IPC), we show that a homolog (open reading frame YDR072c), termed Ipt1 (inositolphosphotransferase 1) is necessary for synthesis of mannose-(inositol-P)2-ceramide (M(IP)2C), the most abundant and complex sphingolipid in S. cerevisiae. This conclusion is based upon analysis of an ipt1-deletion strain, which fails to accumulate M(IP)2C and instead accumulates increased amounts of the precursor mannose-inositol-P-ceramide. The mutant also fails to incorporate radioactive precursors into M(IP)2C, and membranes prepared from it do not incorporate [3H-inositol]phosphatidylinositol into M(IP)2C, indicating a lack of M(IP)2C synthase activity (putatively phosphatidylinositol:mannose-inositol-P-ceramide phosphoinositol transferase). M(IP)2C synthase activity is inhibited in the micromolar range by aureobasidin A, but drug sensitivity is over 1000-fold lower than reported for IPC synthase activity. An ipt1-deletion mutant has no severe phenotypic effects but is slightly more resistant to growth inhibition by calcium ions. Identification of the IPT1 gene should be helpful in determining the function of the M(IP)2C sphingolipid and in determining the catalytic mechanism of IPC and M(IP)2C synthases.

Identification of a Saccharomyces gene, LCB3, necessary for incorporation of exogenous long chain bases into sphingolipids.

To identify genes necessary for sphingolipid synthesis in Saccharomyces cerevisiae we developed a procedure to enrich for mutants unable to incorporate exogenous long chain base into sphingolipids. We show here that a mutant strain, AG84-3, isolated by using the enrichment procedure, makes sphingolipids from endogenously synthesized but not from exogenously supplied long chain base. A gene termed LCB3 (YJL134W, GenBank designation X87371x21), which complements the long chain base utilization defect of strain AG84-3, was isolated from a genomic DNA library. The gene is predicted to encode a protein with multiple membrane-spanning domains and a COOH-terminal glycosylphosphatidylinositiol cleavage/attachment site. Deletion of the lcb3 gene in a wild type genetic background reduces the rate of exogenous long chain base incorporation into sphingolipids and makes the host strain more resistant to growth inhibition by long chain bases. Only one protein in current data bases, the S. cerevisiae open-reading frame YKR053C, whose function is unknown, shows homology to the Lcb3 protein. The two proteins are not, however, functional homologs because deletion of the YKR053C open reading frame does not impair long chain base utilization or enhance resistance of cells to growth inhibition by long chain bases. Based upon these data we hypothesize that the Lcb3 protein is a plasma membrane transporter capable of transporting sphingoid long chain bases into cells. It is the first candidate for such a transporter and the first member of what appears to be a new class of membrane-bound proteins.

Sphingolipid synthesis as a target for antifungal drugs. Complementation of the inositol phosphorylceramide synthase defect in a mutant strain of Saccharomyces cerevisiae by the AUR1 gene.

We have identified a Saccharomyces cerevisiae gene necessary for the step in sphingolipid synthesis in which inositol phosphate is added to ceramide to form inositol-P-ceramide, a reaction catalyzed by phosphatidylinositol:ceramide phosphoinositol transferase (IPC synthase). This step should be an effective target for antifungal drugs. A key element in our experiments was the development of a procedure for isolating mutants defective in steps in sphingolipid synthesis downstream from the first step including a mutant defective in IPC synthase. An IPC synthase defect is supported by data showing a failure of the mutant strain to incorporate radioactive inositol or N-acetylsphinganine into sphingolipids and, by using an improved assay, a demonstration that the mutant strain lacks enzyme activity. Furthermore, the mutant accumulates ceramide when fed exogenous phytosphingosine as expected for a strain lacking IPC synthase activity. Ceramide accumulation is accompanied by cell death, suggesting the presence of a ceramide-activated death response in yeast. A gene, AUR1 (YKL004w), that complements the IPC synthase defect and restores enzyme activity and sphingolipid synthesis was isolated. Mutations in AUR1 had been shown previously to give resistance to the antifungal drug aureobasidin A, leading us to predict that the drug should inhibit IPC synthase activity. Our data show that the drug is a potent inhibitor of IPC synthase with an IC50 of about 0.2 nM. Fungal pathogens are an increasing threat to human health. Now that IPC synthase has been shown to be the target for aureobasidin A, it should be possible to develop high throughput screens to identify new inhibitors of IPC synthase to combat fungal diseases.

A mammalian homolog of the yeast LCB1 encodes a component of serine palmitoyltransferase, the enzyme catalyzing the first step in sphingolipid synthesis.

Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the initial step dedicated to sphingolipid biosynthesis and is thought to be a key enzyme for regulating cellular sphingolipid content. For SPT activity, the yeast Saccharomyces cerevisiae requires two genes, LCB1 and LCB2. We isolated mammalian LCB1 cDNA homologs from mouse and Chinese hamster ovary (CHO) cells and an LCB2 cDNA homolog from CHO cells. The mammalian LCB1 proteins are predicted to have about 35% amino acid identity to the yeast Lcb1 protein, whereas the CHO LCB2 protein is predicted to have about 40% amino acid identity to the yeast Lcb2 protein. Northern blot analysis of mRNA isolated from various mouse tissues revealed that the tissue distribution of both LCB1 and LCB2 messengers followed a similar pattern. Transfection of an SPT-defective CHO mutant strain with a CHO LCB1-expressing plasmid restored both SPT activity and de novo sphingolipid synthesis to the wild type levels, whereas transfection of the mutant strain with a CHO LCB2-expressing plasmid did not exhibit any recovery effects, indicating that the SPT defect in the mutant cells is specifically complemented by the CHO LCB1 homolog. Furthermore, when the SPT-defective mutant cells were transfected with a plasmid encoding a His6-tagged CHO LCB1 protein, SPT activity bound to a Ni2+-immobilized resin. These results indicate that the CHO LCB1 homolog encodes a component of SPT.

Sphingolipid synthesis: identification and characterization of mammalian cDNAs encoding the Lcb2 subunit of serine palmitoyltransferase.

Synthesis of the ceramide portion of sphingolipids in animals has been hypothesized to be tightly regulated thereby controlling the rate of de novo sphingolipid formation. Regulation is predicted to occur at the first and committed biosynthetic step catalyzed by serine palmitoyltransferase (SPT, EC 2.3.1.50). This hypothesis remains unproven because SPT has been refractory to purification and subsequent characterization. To begin to test this hypothesis we have used a genetic strategy to isolate LCB2 homologs from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe and a cDNA homolog from humans and mice. Identity is supported by overall amino acid sequence similarity between the predicted proteins and the known Saccharomyces cerevisiae Lcb2 protein. In addition, a motif of 56 residues from the human protein functionally substituted for the corresponding region of the S. cerevisiae Lcb2 protein. The 56 residue motif was found to be unique to Lcb2 proteins. Likewise, the base sequence encoding it is unique to the human genome. Finally, a peptide sequence in the motif is known to be part of the catalytic domain of all members of the aminolevulinate synthase superfamily of proteins of which Lcb2 is a member. These data argue that this motif is part of the catalytic domain of SPT and is a signature of Lcb2 proteins. The mammalian LCB2 cDNAs provide valuable reagents for studying the Lcb2 subunit of SPT and for studying how ceramide synthesis is regulated.

The LCB2 gene of Saccharomyces and the related LCB1 gene encode subunits of serine palmitoyltransferase, the initial enzyme in sphingolipid synthesis.

The first and committed step in synthesis of the ceramide moiety of sphingolipids is catalyzed by serine palmitoyltransferase (EC 2.3.1.50), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. This step is thought to be tightly regulated to control the synthesis of sphingolipids, but data supporting this hypothesis are lacking mainly because the enzyme has resisted purification and consequent characterization. Rather than attempting to purify the enzyme from normal cells, we have taken a different tack and opted to try and overproduce the enzyme to facilitate its purification. Here we demonstrate that overproduction in Saccharomyces cerevisiae requires expression of LCB1, a previously isolated yeast gene, and LCB2, the isolation and characterization of which we describe. Several lines of evidence argue that both genes encode subunits of the enzyme; however, biochemical evidence will be needed to substantiate this hypothesis. Although overproduction was modest, 2- to 4-fold, it should now be possible to devise improved overproduction vectors for yeast or other host organisms.

A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that resembles an Escherichia coli fatty acyltransferase.

Saccharomyces cerevisiae normally requires sphingolipid biosynthesis for growth; however, mutant strains lacking sphingolipids have been isolated by suppression of a genetic defect in sphingolipid long chain base biosynthesis. To begin to understand the nature of the suppressor(s) we isolated and characterized a suppressor gene, SLC1 (sphingolipid compensation). DNA sequence analysis showed that the wild type SLC1 allele differs from the suppressor allele by a single nucleotide which changes Gln-44 in the predicted wild type protein to Leu4-4 in the predicted SLC1-1 suppressor protein. The predicted SLC1 protein sequence is homologous to the 1-acyl-sn-glycerol-3-phosphate acyltransferase of Escherichia coli encoded by the plsC gene. The homology extends to function as well since the SLC1 gene complements the growth defect in an E. coli strain mutated in plsC. These results suggest that the SLC1 protein has a fatty acyltransferase activity. SLC1 thus may be the first eucaryotic sn2-acylglyceride fatty acyltransferase gene to be cloned. SLC strains grown in the absence of long chain base make novel phosphatidylinositol derivatives (Lester, R. L., Wells, G. B., Oxford, G., and Dickson, R. C. (1993) J. Biol. Chem. 268, 845-856) having a C26 fatty acid at the sn-2 position and the same polar head groups as normal sphingolipids. We postulate that the SLC1 suppressor allele encodes a variant enzyme with an altered substrate specificity that enables it to use a C26 in place of a C16/18 fatty acid precursor to acylate the sn-2 position of inositol-containing glycerolipids.

Autoregulation of GAL4 transcription is essential for rapid growth of Kluyveromyces lactis on lactose and galactose.

Transcriptional induction of genes in the lactose-galactose regulon of the yeast Kluyveromyces lactis requires the GAL4 transcription activator protein. Previous data indicated that the concentration of GAL4 was tightly regulated under basal, inducing, and glucose repressing conditions but the mechanisms were unknown. In this paper we demonstrate that transcription of the GAL4 gene (KI-GAL4) increases 3- to 4-fold during induction of the regulon. This increase requires a KI-GAL4 binding site, UASG, in front of the KI-GAL4 gene, indicating that the KI-GAL4 protein autoregulates transcription of its own gene. Our data demonstrate that the autoregulatory circuit is essential for full induction of the lactose-galactose regulon and, hence, for rapid growth on lactose or galactose. Other data indicate that basal transcription of the KI-GAL4 gene is governed by unidentified promoter elements. The existence of the autoregulatory circuit reveals an important difference between the lactose-galactose regulon and its homologue in Saccharomyces cerevisiae, the melibiose-galactose regulon. This difference may have evolved in response to different selective pressures encountered by the two organisms.

Cloning and mapping of fourteen different DNA fragments containing Aspergillus nidulans 5S rRNA genes.

Fourteen different plasmids hybridizing to Aspergillus nidulans 5S rRNA were isolated from a gene bank obtained after cloning Sau3A partial digests of A. nidulans DNA in a yeast--Escherichia coli vector, pBB29. The restriction maps of these plasmids were determined. The size of the cloned fragments was 2.7-9.5 kb, 12 of the plasmids were found to code for single 5S rRNA genes and 2 coded for 2 genes. No similarity of the sequences surrounding the 5S rRNA genes was found by restriction mapping.

Organization of the ribosomal RNA gene cluster in Aspergillus nidulans.

DNA coding for ribosomal RNA in Aspergillus nidulans was found to consist of a unit 7.8 kb in size which is tandemly repeated in the genome and codes for 5.8S, 18S and 26S rRNA. The repeat unit has been cloned, and its restriction map and the location of the individual rRNA coding sequences within the unit have been established.