M. Marek Nagiec

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) andLCB5 (YLR260w), which encode LCB kinase activity. This conclusion is based upon the synthesis of LCB kinase activity in Escherichia coli expressing eitherLCB 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.

Synthesis of Mannose-(inositol-P)2-ceramide, the Major Sphingolipid in Saccharomyces cerevisiae, Requires the IPT1 (YDR072c) Gene

Knowledge of the Saccharomyces cerevisiaegenes 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 frameYDR072c), termed Ipt1 (inositolphosphotransferase1) 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 thelcb3 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 theYKR053C 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 : 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.

[1] Serine palmitoyltransferase

Publisher Summary The committed step in de novo sphingolipid synthesis begins with the condensation of L -serine and palmitoyl-CoA to produce a C 18 carbon unit, D-3-ketosphinganine, or 3-ketodihydrosphingosine (D-2-amino-l-hydroxyoctadecan-3-one). This reaction, catalyzed by serine palmitoyltransferase (SPT), EC 2.3.1.50, also called “3-ketosphinganine synthase,” was first demonstrated in cell-free extracts made from the yeast Hansenula ciferrii. Shortly thereafter, its existence was shown in extracts prepared from rat liver and mouse brain. Snell and co-workers recognized that the enzyme requires pyridoxal phosphate for activity. The assay of SPT activity is based on the conversion of water-soluble [ 3 H] serine to the chloroform-soluble product, 3-ketosphinganine. The most widely used format of this assay, Protocol I, is based on the original work of Williams et al. and on recent modifications, is Protocol II is a modified format that employs an alternative extraction procedure developed by Wells and Lester (unpublished results) and is used routinely for assay of SPT activity in S. cerevisiae.

Functional characterization of the promoter for the mouse SPTLC2 gene, which encodes subunit 2 of serine palmitoyltransferase.

A series of luciferase reporter constructs was prepared from a 1035‐bp fragment of mouse genomic DNA flanking the 5′‐coding sequence for the SPTLC2 subunit of serine palmitoyltransferase, the initial enzyme of de novo sphingolipid biosynthesis. The full‐length DNA fragment promoted strong reporter gene expression in NIH3T3 cells while deletion and site‐directed mutagenesis indicated that the proximal 335 bp contain initiator and downstream promoter elements, two proximal GC boxes that appear to stimulate transcription in a cooperative manner, and several additional elements whose activity cannot be accounted for by known factor binding sites. These findings provide insight into the control mechanisms for transcription of mammalian SPTLC2.