The existence of auxotrophic mutants of Saccharomyces cerevisiae having an absolute requirement for the long-chain base (lcb) component of sphingolipids suggests that sphingolipids are crucial for viability and growth. One mutant, termed the lcbl-l mutant, lacks the activity of serine palmitoyltransferase, the first enzyme in the pathway for long-chain base synthesis. Here, we present evidence that LCBI has been molecularly cloned. The size of the LCBJ transcript, the direction of transcription, and transcription initiation sites were determined. In addition, the coding region and its 5' and 3' flanking regions were sequenced. Analysis of the DNA sequence revealed a single open reading frame of 1,674 nucleotides, encoding a predicted peptide of 558 amino acids. The hydropathy profile of the predicted peptide suggests a hydrophobic, globular, membrane-associated protein with two potential transmembrane helices. Comparison of the predicted amino acid sequence to known protein sequences revealed homology to 5-aminolevulinic acid synthase and to 2-amino-3-ketobutyrate coenzyme A ligase. These homologies, the similarity of the chemical reactions catalyzed by the three enzymes, and the finding that LCB1 restores serine palmitoyltransferase activity to an lcbl-defective strain indicate that serine palmitoyltransferase or a subunit of the enzyme is the most likely product of LCB1. Homology of the LCB1 predicted protein to the Escherichia coli biotin synthetase was also observed, but the biological significance of this observation is not clear. A role for sphingolipids in sporulation is implicated by our finding that diploids homozygous for kcbl failed to sporulate.Sphingolipids are membrane components found in animals (13), higher plants (18), and fungi (5); they are rarely present in procaryotes (20). In spite of much effort, it has been difficult to understand the exact biological role(s) of sphingolipids and their mode of action at the molecular level. In animals, sphingolipids are thought to play a role in such general cellular events as cell-to-cell recognition, regulation of cell growth, and differentiation (13,19). Sphingolipids have been shown to promote a variety of specific biological activities (for a review, see reference 15). For diseases such as cancer (14), there are changes in the cellular concentration and composition of sphingolipids, but the relationship of these changes to the disease state is unclear. Recently it has been suggested that long-chain bases, such as the sphingolipid precursor sphingosine, and the breakdown products of sphingolipids, lysosphingolipids, may have important biological functions (for a review, see reference 15).Saccharomyces cerevisiae contains a small and unique set of sphingolipids with the compositions inositol-p-ceramide, mannose-inositol-p-ceramide, and mannose-(inositol-p)2-ceramide (36, 39). These sphingolipids and those in other fungi and plants all contain the inositol phosphorylceramide moiety and phytosphingosine. Animal sphingolipids lack inositol phosphate and i...

A selection method for sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae was devised after observing that strains that require a long-chain base for growth become denser when starved for this substance. Genetic analysis of over 60 such strains indicated only two complementation classes, kcbl and kb2. Mutant strains from each class grew equally well with 3-ketodihydrosphingosine, erythrodihydrosphingosine or threodihydrosphingosine, or phytosphingosine. Since these metabolites represent the first, second, and last components, respectively, of the long-chain-base biosynthetic pathway, it is likely that the LCBJ and LCB2 genes are involved in the first step of long-chain-base synthesis. The results of long-chain-base starvation in the Lcb-strains suggest that one or more sphingolipids have a vital role in S. cerevisiae. Immediate sequelae of long-chain-base starvation were loss of viability, exacerbated in the presence of ao-cyclodextrin, and loss of phosphoinositol sphingolipid synthesis but not phosphatidylinositol synthesis. Loss of viability with long-chainbase starvation could be prevented by also blocking either protein or nucleic acid synthesis. Without a long-chain-base, cell division, dry mass accumulation, and protein synthesis continued at a diminished rate and were further inhibited by the detergent Tergitol. The cell density increase induced by long-chain-base starvation is thus explained as a differential loss of cell division and mass accumulation. Long-chain-base starvation in Lcb-S. cerevisiae and inositol starvation of Inos-S. cerevisiae share common features: an increase in cell density and a loss of cell viability overcome by blocking macromolecular synthesis.

We have begun a biochemical-genetic analysis of the synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae and found evidence for the occurrence of serine palmitoyltransferase (SPT) and 3-ketosphinganine reductase, enzymes that catalyze the initial steps of the pathway in other organisms. SPT activity was demonstrated in vitro with crude membrane preparations from S. cerevisiae as judged by the formation of radiolabeled 3-ketosphinganine from the condensation of palmitoyl-coenzyme A (CoA) with radiolabeled serine. Shorter (C12 and C14) and longer (C18) acyl-CoAs sustain significant SPT activity, a result consistent with the finding of both C18 and C20 long-chain bases in the organism. Three products of the long-chain-base synthetic pathway, 3-ketosphinganine, erythrosphinganine, and phytosphingosine, neither directly inhibited the reaction in vitro nor affected the specific activity of the enzyme when these bases were included in the culture medium ofwild-type cells. Thus, no evidence for either feedback inhibition or repression of enzyme synthesis could be found with these putative effectors. Mutant strains of S. cerevisiae that require a sphingolipid long-chain base for growth fall into two genetic complementation groups, LCB1 and LCB2. Membrane preparations from both kcbl and -b2 mutant strains exhibited negligible SPT activity when tested in vitro.Step 2 of the long-chain-base synthetic pathway was demonstrated by the stereospecific NADPHdependent reduction of 3-ketosphinganine to erythrosphinganine. Membranes isolated from wild-type cells and from an kcbl mutant exhibited substantial 3-ketosphinganine reductase activity. We conclude that the Lcbphenotype of these mutants results from a missing or defective SPT, an activity controlled by both the LCBI and LCB2 genes. These results and earlier work from this laboratory establish that SPT plays an essential role in sphingolipid synthesis in S. cerevisiae.Fungi and plants contain sphingolipids that are distinguished from animal sphingolipids; they contain phosphoinositol as part of their polar head groups with phytosphingosine (PHS) as the major long-chain-base (LCB) component (1,8). Such sphingolipids have been shown to be highly localized in the plasma membrane of Saccharomyces cerevisiae (17). To study the metabolism and function of sphingolipids in S. cerevisiae, we isolated mutant strains, termed Lcb-, auxotrophic for sphingolipid LCBs. The Lcb-strains fell into two genetic complementation groups, termed LCBI and LCB2 (18). Without an appropriate LCB, these strains were unable to grow and synthesize sphingolipid (18, 27) and rapidly lost viability (18); these data suggested one or more vital roles for the yeast sphingolipids.No information is available concerning the initial steps of sphingolipid LCB synthesis in S. cerevisiae. We have characterized the first two steps of LCB synthesis (Fig. 1) as catalyzed by isolated membranes from S. cerevisiae. These in vitro enzymatic studies indicate that step 1, carried out by serine palmitoyltransfe...

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