Sphingolipids function as downstream effectors of a fungal PAQR

Abstract

The Izh2p protein from Saccharomyces cerevisiae belongs to the newly characterized progestin and adipoQ receptor (PAQR) superfamily of receptors whose mechanism of signal transduction is still unknown. Izh2p functions as a receptor for the plant PR-5 defensin osmotin and has pleiotropic effects on cellular biochemistry. One example of this pleiotropy is the Izh2p-dependent repression of FET3, a gene involved in iron-uptake. Although the physiological purpose of FET3 repression by Izh2p is a matter of speculation, it provides a reporter with which to probe the mechanism of signal transduction by this novel class of receptor. Receptors in the PAQR family share sequence similarity with enzymes involved in ceramide metabolism, which led to the hypothesis that sphingolipids are involved in Izh2p-dependent signaling. In this study, we demonstrate that drugs affecting sphingolipid metabolism, such as d-erythro-MAPP and myriocin, inhibit the effect of Izh2p on FET3. We also show that Izh2p causes an increase in steady-state levels of sphingoid base. Moreover, we show that Izh2p-independent increases in sphingoid bases recapitulate the effect of Izh2p on FET3. Finally, our data indicate that the Pkh1p and Pkh2p sphingoid base-sensing kinases are essential components of the Izh2p-dependent signaling pathway. In conclusion, our data indicate that Izh2p produces sphingoid bases and that these bioactive lipids probably function as the second messenger responsible for the effect of Izh2p on FET3.

Fig. 1.

Thaumatin represses FET3 expression via Izh2p. In A and B, cells were grown in iron-limited LIM containing 0.05% galactose to modestly overexpress IZH2 driven by the GAL1 promoter. Cell surface ferroxidase activities are plotted as a percentage of the activity seen in wild-type cells carrying empty expression vector. A, WT (BY4742) or isogenic izh2Δ mutant cells carry either empty expression vector (□, ▪) or a plasmid that overexpresses IZH2 (▵). B, ferroxidase activity generated by the FET3 gene shown as a percentage of activity seen in untreated WT (BY4742) cells carrying the IZH2 overexpression vector. All strains carry the IZH2 overexpression plasmid and were either left untreated (□) or exposed to 500 nM thaumatin (). Error bars indicate ± 1 S.E. (n = 3). ▵, isogenic strains lacking particular genes.

Fig. 2.

Hydropathy plots of PAQRs receptors and alkaline ceramidases. In A and B, hydropathy plots for individual proteins were generated and aligned (broken lines). An average hydropathy plot for all proteins plotted in A and B was generated (solid line). The locations of conserved motifs (designated by a circled letter) are shown with letters, and the predicted TMs are numbered. The sequences used to generate these figures are listed in the Supplemental Data. A, various fungal members of the PAQR family. B, various members of the alkaline ceramidase family. C, predicted topology of the PAQR and alkaline ceramidase families with the locations and consensus sequences of the three highly conserved motifs. D, WT (BY4742) and isogenic mutant cells were grown in iron-limited LIM containing 2% galactose to overexpress YPC1 driven by the GAL1 promoter. β-Galactosidase activity generated by the FET3-lacZ reporter is shown as a percentage of activity seen in wild-type cells carrying empty expression vector. ▵, strains lacking particular genes.

Fig. 3.

Involvement of sphingoid bases in Izh2p-dependent signaling. Cells were grown in iron-deficient LIM containing 2% galactose to induce GAL1 driven genes. A, Steady-state levels of phytosphingosine in WT (BY4742) cells carrying either empty expression vector, the YPC1 overexpression vector, or the IZH2 overexpression vector. ^*, p value that is statistically significant at the 95% confidence interval. Broken lines indicate the populations compared using the t tests. Error bars indicate ± 1 S.E. (n = 2). B, the addition of 100 μM exogenous PHS or stearylamine to WT (BY4742) cells carrying empty expression vector represses FET3-lacZ. Untreated cells were actually treated with an equivalent volume of ethanol to control for vehicle effects. Error bars indicate ± 1 S.E. (n = 3). C, the repression of FET3-lacZ caused by treatment of cells with 100 μM PHS was alleviated in tpk2Δ, nrg1Δ, and nrg2Δ mutant strains isogenic to BY4742 WT. Error bars indicate ± 1 S.E. (n = 3). ▵, strains lacking particular genes. D, FB₁ and N,N-DMS repress FET3-lacZ in a dose-dependent manner in WT (BY4742) cells carrying empty expression vector. Error bars indicate ± 1 S.E. (n = 3).

Fig. 4.

Izh2p-dependent signaling does not require endogenous alkaline ceramidases. A, steady-state accumulation of phytosphingosine in ypc1Δydc1Δ double-mutant cells isogenic to WT (W303-1A) carrying either empty expression vector or the YPC1 and IZH2 overexpression plasmids grown in synthetic defined medium with 2% galactose. ^*, p value that is statistically significant at the 95% confidence interval. Broken lines indicate the populations compared using the t tests. Error bars indicate ± 1 S.E. (n = 2). B, steady-state accumulation of phytosphingosine in ypc1Δydc1Δ mutant cells isogenic to WT (W303-1A) carrying either empty expression vector or the IZH2 overexpression plasmid grown in synthetic defined medium with 2% galactose. Cells either were left untreated or were treated for 30 min with 100 μM FB₁ or 1 μM myriocin. Error bars indicate ± 1 S.E. (n = 2). C, β-galactosidase activity from FET3-lacZ is plotted as a percentage of activity seen in untreated wild-type cells carrying empty expression vector as a control. WT (BY4742) cells were grown in iron-deficient LIM containing 2% galactose. The effect of 1-h treatment with myriocin on lacZ activity in wild-type cells carrying either empty expression vector (squares), the IZH2 overexpression plasmid (circles), or the YPC1 overexpression plasmid (triangles). Control cells carrying empty expression vector were also treated with 100 μM PHS (□) or 1 mM Fe³⁺ (▪). Error bars indicate ± 1 S.E. (n = 3).

Fig. 5.

Possible ceramidase function for Izh2p. A, β-galactosidase activity generated by the FET3-lacZ reporter is shown as a percentage of activity seen in untreated WT (BY4742) cells carrying the empty expression vector. Cells were grown in iron-deficient LIM containing either 2% galactose (open and closed symbols) or 0.05% galactose + 500 nM thaumatin (gray symbols). The effect of treatment with d-erythro-MAPP on lacZ activity in wild-type cells carrying either empty expression vector (squares), the IZH2 overexpression plasmid (diamonds), or the YPC1 overexpression plasmid (triangles). Controls show that d-erythro-MAPP has no effect on FET3-lacZ repression caused by either treatment with 100 μM PHS (+) or 1 mM Fe³⁺ (▪). Error bars indicate ± 1 S.E. (n = 3). B, growth curves of yor1Δ mutant cells isogenic to WT (BY4742) carrying either empty expression vector (squares) or the IZH2 (diamonds) and YPC1 (triangles) overexpression plasmids. Cells were either left untreated (gray symbols) or exposed to 100 μM FB₁ (open symbols). Error bars indicate ± 1 S.E. (n = 3).

Fig. 6.

Yeast phosphoinositide-dependent kinases are involved in Izh2p-dependent signaling. A, β-galactosidase activity from FET3-lacZ is plotted as a percentage of activity seen in untreated WT cells carrying empty expression vector as a control. Cells were grown in iron-deficient LIM containing 2% galactose to induce GAL1-driven genes. lacZ activity in WT (BY4742) and pkh1Δ and pkh2Δ isogenic mutant strains carrying either empty expression vector (dotted open bars), the IZH2 overexpression plasmid (open bars), or the YPC1 overexpression plasmid (gray bars). Vector control was also treated with 100 μM PHS (closed bars). Error bars indicate ± 1 S.E. (n = 3). B, β-galactosidase activity from FET3-lacZ is plotted as a percentage of activity seen in untreated WT cells carrying empty expression vector as a control. WT (BY4742) and isogenic mutant cells were grown in iron-deficient LIM containing 0.05% galactose to modestly induce GAL1-driven genes. Cells were treated with 500 nM thaumatin. Error bars indicate ± 1 S.E. (n = 3). ▵, strains lacking particular genes.

Fig. 7.

A model for Izh2p-dependent signaling in yeast cells. Solid lines indicate that a particular molecule or protein acting upon another. Broken lines indicate the effects. Arrows indicate positive effects, whereas bars indicate negative effects.