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.

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.