Yeast strains used in this study.

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

Kes1 regulates Pma1 localization.

<p>SEY6210-<i>PMA1</i>-dsRFP (wild type) and SEY6210-<i>PMA1</i>-dsRFP <i>kes1</i>Δ (CMY306) cells were grown to mid-logarithmic phase in SC medium at 25°C and visualized using a RFP filter. For quantification, 150 and 120 cells were counted for wild type and <i>kes1</i>Δ cells, respectively, over three different experiments to determine the percentage of cells with Pma1-RFP intracellular accumulation (6% for wild type and 81% for <i>kes1</i>Δ). Representative images are shown.</p

Kes1 enhances complex sphingolipid metabolism.

<p>(A) Phosphoinositides plays a central role in the synthesis of sphingolipids in yeast. Gene names are shown in italics. Metabolic intermediates and complex sphingolipids are shown in bold font. Inactivation of <i>KES1</i> inhibits the synthesis of sphingolipids. (B) Cells were grown to late logarithmic phase and inoculated into medium containing <i>myo</i>-[³H]inositol, grown to mid-logarithmic phase and lipids were extracted and resolved by thin layer chromatography. Radioactivity of the resolved lipids was measured for PI, IPC, MIPC and M(IP)₂C. Values were normalized to cell number and are presented as fold-change over wild type. Data represent mean +/− SE of four independent experiments performed in triplicate. *P<0.05 respective to control.</p

Ceramide species is unchanged in cells lacking Kes1.

<p>Lipids were extracted from cells and (A) dihydroceramide (DHCer) and (B) phytoceramide (PHCer) measured by liquid chromatography-mass spectrometry and normalized to total inorganic phosphate.</p

Kes1 affects sphingoid base and ceramide levels.

<p>Sphingolipids were extracted and measured by liquid chromatography-mass spectrometry and normalized to total inorganic phosphate. Total lipid levels representing all chain lengths are given for (A) dihydroceramide (DHC) and phytoceramide (PHC), (B) dihydrosphingosine (DHS) and phytosphingosine (PHS) and (C) dihydrosphingosine-1-phosphate (DHS-1-P) and phytosphingosine-1-phosphate (PHS-1-P). Data are expressed as a mean +/− SE of a minimum of three separate experiments.</p