Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity

Abstract

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...

Sli2 (Ypk1), a Homologue of Mammalian Protein Kinase SGK, Is a Downstream Kinase in the Sphingolipid-Mediated Signaling Pathway of Yeast

“…The effect of ISP-1 on yeast growth was examined since the genes encoding SPT are essential for yeast cell growth (47,53). When yeast cells were treated with ISP-1, cell growth was inhibited after about 5 h (Fig.…”

Sphingolipid long-chain-base auxotrophs of Saccharomyces cerevisiae: genetics, physiology, and a method for their selection

“…This conclusion follows from the finding that 3-KDS, the product of the first step in LCB synthesis, supports the growth of both lcbl and kcb2 strains. In fact, subsequent work (22,23) showed that both lcbl and lcb2 strains have barely detectable levels of serine palmitoyltransferase activity. Furthermore, the LCBI gene has been isolated and its predicted amino acid sequence also strongly suggests that the gene codes for a catalytic segment of serine palmitoyltransferase (2).…”

Cloning of an Alkaline Ceramidase from Saccharomyces cerevisiae

“…On the one hand, ceramide is an essential building block for sphingolipids, whereby sufficient ceramide is needed for cell growth and viability (38,39). On the other hand, ceramide is a stress sensor regulating many cellular responses including heat stress (18), growth arrest (18), and cell death (40).…”