Sphingoid base 1-phosphate phosphatase: a key regulator of sphingolipid metabolism and stress response

Abstract

The sphingolipid metabolites ceramide and sphingosine-1-phosphate are second messengers with opposing roles in mammalian cell growth arrest and survival; their relative cellular level has been proposed to be a rheostat that determines the fate of cells. This report demonstrates that this rheostat is an evolutionarily conserved stress-regulatory mechanism that influences growth and survival of yeast. Although the role of sphingosine-1-phosphate in yeast was not previously examined, accumulation of ceramide has been shown to induce G1 arrest and cell death. We now have identified a gene in Saccharomyces cerevisiae, LBP1, that regulates the levels of phosphorylated sphingoid bases and ceramide. LBP1 was cloned from a yeast mutant that accumulated phosphorylated long-chain sphingoid bases and diverted sphingoid base intermediates from sphingolipid pathways to glycerophospholipid biosynthesis. LBP1 and its homolog, LBP2, encode very hydrophobic proteins that contain a novel-conserved sequence motif for lipid phosphatases, and both have long-chain sphingoid base phosphate phosphatase activity. In vitro characterization of Lbp1p shows that this phosphatase is Mg2+-independent with high specificity for phosphorylated long-chain bases, phytosphingosine and sphingosine. The deletion of LBP1 results in the accumulation of phosphorylated long-chain sphingoid bases and reduced ceramide levels. Moreover, deletion of LBP1 and LBP2 results in dramatically enhanced survival upon severe heat shock. Thus, these phosphatases play a previously unappreciated role in regulating ceramide and phosphorylated sphingoid base levels in yeast, and they modulate stress responses through sphingolipid metabolites in a manner that is reminiscent of their effects on mammalian cells.

Figure 1

Pathways of sphingolipid and glycerophospholipid metabolism in S. cerevisiae. DG, diacylglycerol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol; KDS, ketodihydrosphingosine; DHS, dihydrosphingosine; PHS, phytosphingosine; IPC, inositol phosphorylceramide; MIPC, mannose inositol phosphorylceramide; and M(IP)₂C, mannose diinositol diphosphorylceramide.

Figure 2

(A) LBP1 complements hypersensitivity of lbp1–1 to australifungin. Wild-type strain W303-1A/YCp (○) or lbp1–1 mutant transformed with control vector (•), LBP1 on low copy YCp vector (▪), LBP1 on multicopy YEp vector (▴), or LBP2 on multicopy YEp vector (X) were tested for their sensitivity to australifungin. The data are expressed as percent of growth of W303-1A. (B) Δlbp1 and lbp1–1 are hypersensitive to australifungin. The sensitivity of lbp1–1 (•), Δlbp1 (□), Δlbp2 (○) haploid mutants, and Δlbp1/LBP1 heterozygote (▴) to australifungin was compared with the sensitivity of wild-type diploid strain YSP047 (▪). (C and D) Δlbp1, but not Δlbp2, suppresses the ethanolamine requirement of Δcho1. Serial dilutions of overnight cultures were spotted onto SC agar plates with (C) or without (D) 10 mM ethanolamine. Strains: 1, W303–1A; 2, Δcho1; 3, Δcho1Δlbp1; 4, Δcho1Δlbp2; and 5, Δcho1Δlbp1Δlbp2.

Figure 3

The product of the LBP1 gene is an LCBP phosphatase. (A) LCBP-phosphatase activity was measured in wild-type (WT) W303-1A, Δlbp1, Δlbp2, and W303 cells carrying LBP1 or LBP2 on multicopy YEp plasmids by using phytosphingosine-1-³²P as substrate. (B) Effects of lipids and phosphatase inhibitors on LCBP-phosphatase activity of wild-type W303 cells (open bars) and the LBP1 overexpressor strain (hatched bars). The following agents were used: Triton X-100, 0.05%; hexadecylphosphate (100 μM); tartrate (10 mM); propanolol (4 mM); N-ethylmaleimide (NEM, 5 mM); phosphoethanolamine (10 mM); phosphoserine (10 mM); pyrophosphate (10 mM); glycerol 3-phosphate (6 mM); and NaF (10 mM). (C) Inhibition of LCBP-phosphatase activity by 100 μM concentrations of the indicated lipids in membrane fractions from the LBP1 overexpressor strain using phytosphingosine-1-³²P (open bars) or sphingosine-1-³²P (hatched bars) as substrates.

Figure 4

Δlbp1 accumulates LCBP, has reduced levels of ceramide, and fails to incorporate exogenous dihydrosphingosine into sphingolipids. Strains W3031A (lanes 1, 3, and 6), lbp1–1 (lane 2), Δlbp 1 (lanes 4 and 7), or Δlbp2 (lanes 5 and 8) were labeled with the sphingolipid precursors: [³H]palmitate (lanes 1 and 2), [³H]serine (lanes 3–5), or [³H]dihydrosphingosine (lanes 6–8), and the lipids were extracted and processed as described in Materials and Methods and separated by using TLC. Migration of standards is indicated: CER, ceramide; GPE, glycerophosphate ethanolamine; GPS, glycerophosphate serine; GPC, glycerophosphate choline; all other standards as described in Fig. 1.

Figure 5

(A, B) Δlbp mutants are protected from loss of viability upon heat shock. (A) Yeast strains were grown in SC medium and heat-shocked at 50°C for the indicated times, and then cell growth after 24 h was determined; W303 (□), Δlbp1 (▴), and W303 cells carrying LBP1 on multicopy plasmids (•). (B) Strains were grown overnight in SC (Left) or YPD (Right), and heat-shocked for 45 min, and viability was determined. (C) Δlbp2 has reduced LCBP phosphatase activity in YPD media. W3031A (WT), Δlbp1, Δlbp2, and Δlbp1Δlbp2 strains were grown in YPD media to OD₆₀₀ of 11, and LCBP phosphatase activity was measured, as described in Materials and Methods, using sphingosine-1-³²P as substrate.