A Yeast Mutant Deleted of GPH1 Bears Defects in Lipid Metabolism

Abstract

In a previous study we demonstrated up-regulation of the yeast GPH1 gene under conditions of phosphatidylethanolamine (PE) depletion caused by deletion of the mitochondrial (M) phosphatidylserine decarboxylase 1 (PSD1) (Gsell et al., 2013, PLoS One. 8(10):e77380. doi: 10.1371/journal.pone.0077380). Gph1p has originally been identified as a glycogen phosphorylase catalyzing degradation of glycogen to glucose in the stationary growth phase of the yeast. Here we show that deletion of this gene also causes decreased levels of phosphatidylcholine (PC), triacylglycerols and steryl esters. Depletion of the two non-polar lipids in a Δgph1 strain leads to lack of lipid droplets, and decrease of the PC level results in instability of the plasma membrane. In vivo labeling experiments revealed that formation of PC via both pathways of biosynthesis, the cytidine diphosphate (CDP)-choline and the methylation route, is negatively affected by a Δgph1 mutation, although expression of genes involved is not down regulated. Altogether, Gph1p besides its function as a glycogen mobilizing enzyme appears to play a regulatory role in yeast lipid metabolism.

Fig 1. Calcofluor White sensitivity of Δgph1, Δpsd1 and Δglc3 deletion mutants.

Wild type BY4741 and mutant strains as indicated were grown on YPD plates and on YPD plates containing 20 μg/ml Calcofluor White (CFW).

Fig 2. Total amount of phospholipids in cell free homogenate from wild type and the Δgph1 deletion mutant.

Cells were grown on YPD to the early stationary growth phase at 30°C and disrupted with the aid of glass beads. Lipids were extracted with chloroform/methanol (2:1; v/v). Black bar, wild type BY4741; grey bar, Δgph1 mutant. (A) Total amounts of phospholipids were related to the amounts of protein. (B) Total amounts of phospholipids were related to cell wet weight. Inorganic phosphate was used as standard.

Fig 3. Synthesis of phosphatidylcholine is down regulated in the Δgph1 deletion mutant in vivo.

Black bar, wild type BY4741; grey bar, Δgph1 deletion mutant; (A) The so-called Kennedy pathway (CDP-choline pathways) was analyzed by incorporation of [methyl-¹⁴C]choline chloride into PC. (B) To analyze the methylation pathway of PC, the incorporation of L-[³H]serine into PS, PE and PC was measured. Wild type BY4741 was set at 100%.

Fig 4. Expression levels of PSD1, PSD2, CHO2 and OPI3 in the Δgph1 deletion mutant.

Relative gene expression of PSD1, PSD2, CHO2 and OPI3 in the Δgph1 deletion mutant was measured by RT-qPCR. A Δpsd1 mutant was used as a negative control. Wild type was set at 1. Data are mean values from three independent experiments with the respective deviation.

Fig 5. Glycogen content in the Δgph1, Δpsd1 and Δglc3 deletion mutants.

Strains as indicated were grown on YPD plates. Cell suspensions were spotted at dilutions 1, 1/10, 1/100, 1/1000, 1/10000 and incubated at 30°C for 48 h. Plates were exposed to iodine vapor for 10 min.

Fig 6. Amounts of triacylglycerols and steryl esters are reduced in the Δgph1 deletion mutant.

The wild type BY4741 (black bar) and the Δgph1 deletion mutant (grey bar) were grown aerobically to the early stationary growth phase, and triacylglycerols (TG), diacylglycerols (DG), steryl esters (SE) and ergosterol were quantified. Results were obtained from 3 independent samples with deviations as indicated by the error bars.

Fig 7. Number and size of lipid droplets are decreased in the Δgph1 deletion mutant.

(A) Transmission electron microscopy images of wild type BY4741 and Δgph1. (B) Nile Red staining and fluorescence microscopy of wild type BY4741 and Δgph1. LD: lipid droplets. Scale bar: 2 μm.

Fig 8. Triacylglycerol degradation in the Δgph1 mutant.

Wild type BY4741 (A) and Δgph1 mutant cells (B) were grown to an OD₆₀₀ of 3. Cerulenin was added at time point 0 to a final concentration of 10 μg/μl (grey line; -●-). The black line (-■-) shows the control without cerulenin. Over the time period of TG degradation the amount of glycogen was tested in wild type BY4741 (black bars) and Δgph1 (grey bars). Amounts of glycogen were expressed as μg/OD₆₀₀ unit. Results were obtained from 3 independent samples with deviations as indicated by the error bars.

Fig 9. Fatty acid profile of the Δgph1 mutant.

The distribution of myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0) and oleic acid (C18:1) was analyzed in wild type BY4741 (black bar) and in the Δgph1 strain (grey bar). Results were obtained from 3 independent samples with deviations as indicated by the error bars.