Structural parameters of mitochondria from different mutant strains.

<p>Electron micrographs of the tested strains were obtained as described in the Materials and Methods section. For the analysis of mitochondrial size, circumference and area 70 to 100 mitochondria were tested.</p

Analysis of organelles from wild type, <i>cho2Δopi3Δ</i> and <i>cki1Δdpl1Δeki1Δ</i> yeast strains.

<p>Yeast strains used were grown on YPO media. Data obtained with isolated peroxisomes (lane A), mitochondria (lane B) and endoplasmic reticulum (lane C) are shown. Line 1: Phospholipid pattern expressed as μg of individual phospholipids per mg protein. LP, lysophospholipids; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; CL, cardiolipin; PA, phosphatidic acid. Line 2: PC to PE ratio in different organelles and strains. Line 3: Fatty acid composition of different organelles and strains. Line 4: Ratio of saturated (SFA) to unsaturated (UFA) fatty acids in different organelles and strains. Line 5: Ergosterol (ERG) to phospholipid (PL) ratio in different organelles and strains. Line 6: Anisotropy values obtained with different organelles and strains. For experimental details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135084#sec002" target="_blank">Materials and Methods</a> section. For all experiments two independent biological samples were used which were analyzed 2 to 3 time, each.</p

Transmission electron microscopy of wild type and mutant strains.

<p>Ultrathin sections of chemically fixed <i>S</i>. <i>cerevisiae</i> yeast cells are shown. For experimental details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135084#sec002" target="_blank">Materials and Methods</a> section. Mitochondria are marked with arrows. Wild type (A), <i>cki1Δdpl1Δeki1Δ</i> (B), and <i>cho2Δopi3Δ</i> (C) were grown on YPO to induce formation of peroxisomes.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

A Yeast Mutant Deleted of <i>GPH1</i> Bears Defects in Lipid Metabolism

<div><p>In a previous study we demonstrated up-regulation of the yeast <i>GPH1</i> gene under conditions of phosphatidylethanolamine (PE) depletion caused by deletion of the mitochondrial (M) phosphatidylserine decarboxylase 1 (<i>PSD1</i>) (Gsell et al., 2013, PLoS One. 8(10):e77380. doi: <a href="http://dx.doi.org/10.1371/journal.pone.0077380" target="_blank">10.1371/journal.pone.0077380</a>). 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 Δ<i>gph1</i> strain leads to lack of lipid droplets, and decrease of the PC level results in instability of the plasma membrane. <i>In vivo</i> 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 Δ<i>gph1</i> 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.</p></div

Fatty acids in the homogenate from wild type, <i>cho2Δopi3Δ</i> and <i>cki1Δdpl1Δeki1Δ</i> yeast strains.

<p>Yeast strains used were grown on YPO media, and homogenate samples were prepared after cell disruption. The oleic acid content was quantified as described in the Materials and Methods section.</p

Pathways of phosphatidylcholine biosynthesis in the yeast <i>S</i>. <i>cerevisiae</i>.

<p>Cct1, cholinephosphate cytidylyltransferase; CDP-Cho, cytidine-diphosphocholine, CDP-Etn, cytidine-diphosphoethanolamine; Cho, Choline; Cho2, phosphatidylethanolamine methyltransferase; Cho-P, phosphocholine; Cki1, choline kinase; Cpt1, cholinephosphotransferase; DAG, diacylglycerol; Dpl1, sphingosine phosphate lyase; Ect1, phosphoethanolamine cytidylyltransferase; Eki1, ethanolamine kinase; Ept1, sn-1,2-diacylglycerol ethanolamine- and cholinephosphotranferase; Etn, ethanolamine; Etn-P, phosphoethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PDME, phosphatidyldimethylethanolamine; PMME phosphatidylmonomethylethanolamine; PS, phosphatidylserine; Psd1, phosphatidylserine decarboxylase 1; Psd2, phosphatidylserine decarboxylase 2; Opi3, methylene-fatty-acyl-phospholipid synthase; SAM, S-adenosyl-L-methionine; SL, sphingolipids.</p

Growth phenotype of wild type, <i>cho2Δ</i>, <i>opi3Δ</i>, <i>cho2Δopi3Δ</i> and <i>cki1Δdpl1Δeki1Δ</i> yeast strains.

<p>(A): Drop test on YPD plates (30°C and 37°C); on minimal oleate media containing choline and ethanolamine; and on YPO plates are shown. (B) Growth of liquid cultures on YPO.</p

Quantification of Western blot analysis of subcellular fractions from <i>S</i>. <i>cerevisiae</i>.

<p>ER (endoplasmic reticulum), M (mitochondria) and PX (peroxisomes) were compared to the H (homogenate) of the wild type strain (A), the <i>cki1</i>Δ<i>dpl1</i>Δ<i>eki1</i>Δ (B) and the <i>cho2</i>Δ<i>opi3</i>Δ (C) mutant strains.</p

Expression levels of <i>PSD1</i>, <i>PSD2</i>, <i>CHO2</i> and <i>OPI3</i> in the Δ<i>gph1</i> deletion mutant.

<p>Relative gene expression of <i>PSD1</i>, <i>PSD2</i>, <i>CHO2</i> and <i>OPI3</i> in the Δ<i>gph1</i> deletion mutant was measured by RT-qPCR. A <i>Δpsd1</i> 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.</p