Cloning and Characterization of a Saccharomyces cerevisiae Alkaline Ceramidase with Specificity for Dihydroceramide

Mao, Cungui; Xu, Ruijuan; Bielawska, Alicja; Szulc, Zdzisław M.; Obeid, Lina M.

doi:10.1074/jbc.m003683200

Cited by 138 publications

(148 citation statements)

References 29 publications

Supporting

Mentioning

143

Contrasting

Order By: Relevance

“…In addition, labeled C 2 -dihydroceramide in S. cerevisiae was rapidly internalized, metabolized, and incorporated into complex sphingolipids. 3 Thus, C 2 -PHC is also probably internalized, converted to PHS by ceramidases (23,36), and incorporated into complex sphingolipids. Therefore, C 2 -PHC does not likely cause accumulation of PHS and consequently does not play a role in PHS-mediated growth inhibition.…”

Section: Discussionmentioning

confidence: 99%

Phytosphingosine as a Specific Inhibitor of Growth and Nutrient Import in Saccharomyces cerevisiae

Chung¹,

Mao²,

Heitman³

et al. 2001

Journal of Biological Chemistry

Self Cite

110

View full text Add to dashboard Cite

In this study, we used a combination of pharmacological and genetic approaches to determine which endogenous sphingolipid is the likely mediator of growth inhibition. When cells were treated with exogenous phytosphingosine (PHS, 20 M) or structurally similar or metabolically related molecules, including 3-ketodihydrosphingosine, dihydrosphingosine, C 2 -phytoceramide (PHC), and stearylamine, only PHS inhibited growth. Also, PHS was shown to inhibit uptake of uracil, tryptophan, leucine, and histidine. Again this effect was specific to PHS. Because of the dynamic nature of sphingolipid metabolism, however, it was difficult to conclude that growth inhibition was caused by PHS itself. By using mutant yeast strains defective in various steps in sphingolipid metabolism, we further determined the specificity of PHS. The elo2⌬ strain, which is defective in the conversion of PHS to PHC, was shown to have slower biosynthesis of ceramides and to be hypersensitive to PHS (5 M), suggesting that PHS does not need to be converted to PHC. The lcb4⌬ lcb5⌬ strain is defective in the conversion of PHS to PHS 1-phosphate, and it was as sensitive to PHS as the wild-type strain. The syr2⌬ mutant strain was defective in the conversion of DHS to PHS. Interestingly, this strain was resistant to high concentrations of DHS (40 M) that inhibited the growth of an isogenic wild-type strain, demonstrating that DHS needs to be converted to PHS to inhibit growth. Together, these data demonstrate that the active sphingolipid species that inhibits yeast growth is PHS or a closely related and yet unidentified metabolite.Certain sphingolipid metabolites including ceramide, sphingosine, and sphingosine 1-phosphate have pleiotropic effects on cellular growth and proliferation. The yeast Saccharomyces cerevisiae has emerged as an excellent model system for studying sphingolipid-mediated signal transduction. First, compared with over 300 different kinds of sphingolipids found in mammalian cells, there is only a limited number of sphingolipid species in the yeast, which simplifies lipid analysis (2, 3). Moreover, the basic structure, biosynthesis, and metabolism of sphingolipids are well conserved between mammalian and yeast systems. Second, many yeast genes in the sphingolipid biosynthetic and metabolic pathways have been cloned, providing opportunities for studying the effects of endogenous sphingolipids using genetics tools. Finally, although this is not exclusive to sphingolipid studies, yeast genetics provide excellent tools to identify and characterize components in signal transduction pathways (4 -6).Evidence for conservation of the sphingolipid signaling pathway in yeast comes from several studies. These include demonstrating that D-erythro-ceramide inhibited yeast cell growth in liquid culture and activated a protein phosphatase 2A that could be inhibited by okadaic acid (7). Later, Nickels and Broach (8) showed that ceramide inhibited yeast cell growth by arresting cell cycle at G 1 phase and that ceramide-activated protein phosphatase is com...

show abstract

Section: Discussionmentioning

confidence: 99%

Phytosphingosine as a Specific Inhibitor of Growth and Nutrient Import in Saccharomyces cerevisiae

Chung¹,

Mao²,

Heitman³

et al. 2001

Journal of Biological Chemistry

Self Cite

110

View full text Add to dashboard Cite

show abstract

“…The construct pYES2-CDase/His 9 or the empty vector pYES2 was transformed into the yeast strain ∆ypc1∆ydc1 by the lithium acetate method, as previously described [29]. The yeast double-knockout strain ∆ypc1∆ydc1, which lacks the yeast CDases YDC1p and YPC1p, had been generated previously [6]. The strain containing pYES2-CDase/His 9 was grown and maintained in synthetic complete medium (SC-Ura − ) with the omission of uracil.…”

Section: Expression Of the Human Neutral Cdase In Yeastmentioning

confidence: 99%

“…This enzyme has a pH optimum of 4-5, is glycosylated, has an apparent molecular mass of 50 kDa and it prefers ceramide as a substrate over dhCer (dihydroceramide). Recently, two yeast (Saccharomyces cerevisiae) alkaline CDases, namely YPC1p (phytoCDase) and YDC1p (dihydroCDase), were cloned and characterized [5,6]. YPC1p and YDC1p have pH optima at approx.…”

Section: Introductionmentioning

confidence: 99%

Identification of a novel amidase motif in neutral ceramidase

Galadari

Mao

et al. 2006

Biochemical Journal

Self Cite

View full text Add to dashboard Cite

Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast doubleknockout strain ∆ypc1∆ydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na + and Ca 2+ . Mg 2+ and Mn 2+ were somewhat stimulatory, but Zn 2+ , Cu 2+ and Fe 2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser 354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXG P / X XC. Moreover, mutations of Asp 352 and Cys 362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide.

show abstract

“…These include enzymes like ceramidases that break down ceramide (5,6) and the LCBP lyase that cleaves these phosphorylated lipids into ethanolamine and a long chain aldehyde (7). More recent experiments in the yeast S. cerevisiae have identified a new membrane protein that is thought to efflux LCBs out of the cell.…”

mentioning

confidence: 99%

Long Chain Base Tolerance in Saccharomyces cerevisiae Is Induced by Retrograde Signals from the Mitochondria

Panwar

Moye‐Rowley

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Saccharomyces cerevisiae cells lacking their mitochondrial DNA ( 0 cells) respond to this loss of genetic information by induction of a program of nuclear gene expression called the retrograde response. Expression of genes involved in multidrug resistance and sphingolipid biosynthesis is coordinately induced in 0 cells by the zinc cluster transcription factor Pdr3p. In this report, we identify a membrane protein involved in control of intracellular levels of a sphingolipid precursor as a transcriptional target of the Pdr3p-mediated retrograde response. These sphingolipid precursors are called long chain bases (LCBs) and increased LCB levels are growth inhibitory. This membrane protein has been designated Rsb1p and has previously been shown to act as a LCB transporter protein and to be a component of the endoplasmic reticulum. These earlier studies used an amino-terminal truncated form of Rsb1p. Here we employ a full-length form of Rsb1p and find that this protein is localized to the plasma membrane and is modified by N-linked glycosylation. Two glycosylation sites are present in the Rsb1p and both are required for normal LCB resistance. Mutational analysis of the RSB1 promoter revealed that two Pdr3p binding sites are present and both of these are required for normal retrograde induction of transcription. LCB tolerance is strongly increased in 0 cells but this increase is ablated in 0 rsb1⌬ cells. Together, these data indicate Pdr3p activation of RSB1 transcription is an important feature of the retrograde response allowing normal detoxification of an endogenous sphingolipid precursor.Sphingolipids are important lipid constituents of eukaryotic membranes. Key intermediates in the biosynthesis of sphingolipids are the long chain bases (LCBs).2 In Saccharomyces cerevisiae, these LCBs are dihydrosphingosine and phytosphingosine (PHS) (reviewed in Ref. 1). These LCBs are utilized by ceramide synthase to form ceramide that is a well known bioactive lipid (reviewed in Refs. 2 and 3). However, LCBs and their phosphorylated forms (LCBPs) have also recently been found to serve as potent signaling molecules. LCBs appear to act as inhibitors of proliferation, whereas increased LCBP levels stimulate growth (recently reviewed in Ref. 4). Control of the biological levels of these signaling lipids is an essential feature for ensuring both normal lipid composition of membranes and proper metabolic regulation.The central importance of these signaling and structural lipids is likely to explain the multitude of regulatory mechanisms modulating their levels. These include enzymes like ceramidases that break down ceramide (5, 6) and the LCBP lyase that cleaves these phosphorylated lipids into ethanolamine and a long chain aldehyde (7). More recent experiments in the yeast S. cerevisiae have identified a new membrane protein that is thought to efflux LCBs out of the cell. This protein has been designated Rsb1p (resistant to sphingoid bases) as it was identified as a high-copy suppressor of the PHS hypersensitivity of a dpl1⌬ strain (...

show abstract

Cloning and Characterization of a Saccharomyces cerevisiae Alkaline Ceramidase with Specificity for Dihydroceramide

Cited by 138 publications

References 29 publications

Phytosphingosine as a Specific Inhibitor of Growth and Nutrient Import in Saccharomyces cerevisiae

Phytosphingosine as a Specific Inhibitor of Growth and Nutrient Import in Saccharomyces cerevisiae

Identification of a novel amidase motif in neutral ceramidase

Long Chain Base Tolerance in Saccharomyces cerevisiae Is Induced by Retrograde Signals from the Mitochondria

Contact Info

Product

Resources

About