Robert C. Dickson

Biochemistry, Cell Biology and Molecular Biology of Lipids of Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a powerful experimental system to study biochemical, cell biological and molecular biological aspects of lipid synthesis. Most but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of this unicellular eukaryote have been cloned, and many gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes, turnover and degradation of complex lipids, regulation of lipid biosynthesis, and linkage of lipid metabolism to other cellular processes. Here we summarize current knowledge about lipid biosynthetic pathways in S. cerevisiae and describe the characteristic features of the gene products involved. We focus on recent discoveries in these fields and address questions on the regulation of lipid synthesis, subcellular localization of lipid biosynthetic steps, cross‐talk between organelles during lipid synthesis and subcellular distribution of lipids. Finally, we discuss distinct functions of certain key lipids and their possible roles in cellular processes.

SPHINGOLIPIDS ARE POTENTIAL HEAT STRESS SIGNALS IN SACCHAROMYCES

The ability of organisms to quickly respond to stresses requires the activation of many intracellular signal transduction pathways. The sphingolipid intermediate ceramide is thought to be particularly important for activating and coordinating signaling pathways during mammalian stress responses. Here we present the first evidence that ceramide and other sphingolipid intermediates are signaling molecules in the Saccharomyces cerevisiaeheat stress response. Our data show a 2–3-fold transient increase in the concentration of C18-dihydrosphingosine and C18-phytosphingosine, more than a 100-fold transient increase in C20-dihydrosphingosine and C20-phytosphingosine, and a more stable 2-fold increase in ceramide containing C18-phytosphingosine and a 5-fold increase in ceramide containing C20-phytosphingosine following heat stress. Treatment of cells with dihydrosphingosine activates transcription of the TPS2 gene encoding a subunit of trehalose synthase and causes trehalose, a known thermoprotectant, to accumulate. Dihydrosphingosine induces expression of aSTRE-LacZ reporter gene, showing that the global stress response element, STRE, found in many yeast promoter sequences can be activated by sphingolipid signals. TheTPS2 promoter contains four STREs that may mediate dihydrosphingosine responsiveness. Using genetic and other approaches it should be possible to identify sphingolipid signaling pathways in S. cerevisiae and quantify the importance of each during heat stress.

Sphingolipid functions in Saccharomyces cerevisiae

Recent advances in understanding sphingolipid metabolism and function in Saccharomyces cerevisiae have moved the field from an embryonic, descriptive phase to one more focused on molecular mechanisms. One advance that has aided many experiments has been the uncovering of genes for the biosynthesis and breakdown of sphingolipids. S. cerevisiae seems on the verge of becoming the first organism in which all sphingolipid metabolic genes are identified. Other advances include the demonstration that S. cerevisiae cells have lipid rafts composed of sphingolipids and ergosterol and that specific proteins associate with rafts. Roles for phytosphingosine (PHS) and dihydrosphingosine (DHS) in heat stress continue to be uncovered including regulation of the transient cell cycle arrest, control of putative signaling pathways that govern cell integrity, endocytosis, movement of the cortical actin cytoskeleton and regulation of protein breakdown in the plasma membrane. Other studies suggest roles for sphingolipids in exocytosis, growth regulation and longevity. Finally, some progress has been made in understanding how sphingolipid synthesis is regulated and how sphingolipid levels are maintained.

Initiating oncogenic event determines gene-expression patterns of human breast cancer models

Molecular expression profiling of tumors initiated by transgenic overexpression of c-myc, c-neu, c-ha-ras, polyoma middle T antigen (PyMT) or simian virus 40 T/t antigen (T-ag) targeted to the mouse mammary gland have identified both common and oncogene-specific events associated with tumor formation and progression. The tumors shared great similarities in their gene-expression profiles as compared with the normal mammary gland with an induction of cell-cycle regulators, metabolic regulators, zinc finger proteins, and protein tyrosine phosphatases, along with the suppression of some protein tyrosine kinases. Selection and hierarchical clustering of the most variant genes, however, resulted in separating the mouse models into three groups with distinct oncogene-specific patterns of gene expression. Such an identification of targets specified by particular oncogenes may facilitate development of lesion-specific therapeutics and preclinical testing. Moreover, similarities in gene expression between human breast cancers and the mouse models have been identified, thus providing an important component for the validation of transgenic mammary cancer models.

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.

METABOLISM AND SELECTED FUNCTIONS OF SPHINGOLIPIDS IN THE YEAST SACCHAROMYCES CEREVISIAE

Our knowledge of sphingolipid metabolism and function in Saccharomyces cerevisiae is growing rapidly. Here we discuss the current status of sphingolipid metabolism including recent evidence suggesting that exogenous sphingoid long-chain bases must first be phosphorylated and then dephosphorylated before incorporation into ceramide. Phenotypes of strains defective in sphingolipid metabolism are discussed because they provide hints about the undiscovered functions of sphingolipids and are one of the major reasons for studying this model eukaryote. The long-chain base phosphates, dihydrosphingosine-1-phosphate and phytosphingosine-1-phosphate, have been hypothesized to play roles in heat stress resistance, perhaps acting as signaling molecules. We evaluate the data supporting this hypothesis and suggest future experiments needed to verify it. Finally, we discuss recent clues that may help to reveal how sphingolipid synthesis and total cellular sphingolipid content are regulated.

SYRINGOMYCIN ACTION GENE SYR2 IS ESSENTIAL FOR SPHINGOLIPID 4-HYDROXYLATION IN SACCHAROMYCES CEREVISIAE

The Saccharomyces cerevisiae geneSYR2, necessary for growth inhibition by the cyclic lipodepsipeptide syringomycin E, is shown to be required for 4-hydroxylation of long chain bases in sphingolipid biosynthesis. Four lines of support for this conclusion are presented: (a) the predicted Syr2p shows sequence similarity to diiron-binding membrane enzymes involved in oxygen-dependent modifications of hydrocarbon substrates, (b) yeast strains carrying a disrupted SYR2 allele produced sphingoid long chain bases lacking the 4-hydroxyl group present in wild type strains, (c) 4-hydroxylase activity was increased in microsomes prepared from a SYR2 overexpression strain, and (d) the syringomycin E resistance phenotype of asyr2 mutant strain was suppressed when grown under conditions in which exogenous 4-hydroxysphingoid long chain bases were incorporated into sphingolipids. The syr2 strain produced wild type levels of sphingolipids, substantial levels of hydroxylated very long chain fatty acids, and the full complement of normal yeast sphingolipid head groups. These results show that the SYR2gene is required for the 4-hydroxylation reaction of sphingolipid long chain bases, that this hydroxylation is not essential for growth, and that the 4-hydroxyl group of sphingolipids is necessary for syringomycin E action on yeast.

Direct selection of Saccharomyces cerevisiae resistant to the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-resistance gene of Tn903

We have developed a new procedure for selecting yeast transformants without the need for complementing auxotrophic markers. The procedure is based on resistance to antibiotic G418 imparted to transformants by recombinant DNA vectors. We constructed several Escherichia coli-yeast shuttle vectors containing the kanamycin (G418)-resistance gene of Tn903, plus several yeast genes making dual selections possible. The efficiency for selecting G418-resistant transformants was dependent upon several factors including the composition of the growth medium and the time at which G418 selective pressure was administered. Media which contained levels of salts found in yeast nitrogen base rendered cells partially to completely resistant to G418 and could not be used for selecting G418-resistant transformants. On the other hand, untransformed cells remained sensitive to G418 when grown on YEPD medium thus allowing selection of G418-resistant transformants. A lag phase of 12 to 18 h, following growth at 30 degrees C, was required prior to administration of G418 to achieve maximal transformation frequency. Transformation frequencies ranged from 100 to 700 per micrograms of DNA and varied with the vector and strain used. The kanamycin gene imparted resistance to G418 in either the episomally or chromosomally integrated state. The gene was highly stable in the integrated state, even without selective pressure. The utility of the procedure was demonstrated by selecting transformants of four different strains of Saccharomyces cerevisiae and by cloning autonomous replication sequences (ARS) from the yeast Kluyveromyces lactis. We believe that this or related procedures could be used to develop transformation systems for many eukaryotic and prokaryotic cells for which no transformation procedure is available.

Pil1p and Lsp1p Negatively Regulate the 3-Phosphoinositide-dependent Protein Kinase-like Kinase Pkh1p and Downstream Signaling Pathways Pkc1p and Ypk1p

The Saccharomyces cerevisiae homologs, Pkh1/2p, of the mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1) regulate the Pkc1-MAP kinase cascade and the partially parallel Ypk1/2p pathway(s) that control growth and cell integrity. Mammalian PDK1 is regulated by 3-phosphoinositides, whereas Pkh1/2p are regulated by sphingolipid long-chain bases (LCBs). Recently Pkh1/2p were found to complex with two related proteins, Pil1p (Ygr086) and Lsp1p (Ypl004). Because these two proteins are not related to any known protein we sought to characterize their functions. We show that Pkh1p phosphorylates both proteins in vitro in a reaction that is only weakly regulated by LCBs. In contrast, LCBs inhibit phosphorylation of Pil1p by Pkh2p, whereas LCBs stimulate phosphorylation of Lsp1p by Pkh2p. We find that Pil1p and Lsp1p down-regulate resistance to heat stress and, specifically, that they down-regulate the activity of the Pkc1p-MAP and Ypk1p pathways during heat stress. Pil1p and Lsp1p are thus the first proteins identified as regulators of Pkh1/2p. An unexpected finding was that the level of Ypk1p is greatly reduced in pkc1Δ cells, indicating that Pkc1p controls the level of Ypk1p. Homologs of Pil1p and Lsp1p are widespread in nature, and our results suggest that they may be negative regulators of PDK-like protein kinases and their downstream cellular pathways that control cell growth and survival.