Effect of temperature on Sec14 and Sec14 ^G266D protein levels.

<p><i>A</i>, the <i>sec14^ts</i> cells were transformed with a plasmid expressing Sec14 containing an N-terminal T7 epitope, untagged Sec14, or empty vector. <i>SEC14</i> expression was driven by the constitutive <i>GPD1</i> promoter. Cells were grown in solution at 25°C to mid-logarithmic phase, and serial dilutions of identical numbers of cells were spotted onto plates and incubated at 37°C for two days. <i>B</i>, cells expressing T7-Sec14 or Sec14^G266D were grown to mid-logarithmic phase at 25°C, with a subset shifted to 37°C for 2 hrs. Cells were disrupted by three passes through a French press and membranes were separated from soluble proteins by differential centrifugation. Proteins in each fraction were separated by SDS-PAGE, transferred to PVDF membrane, and western blots were performed. In the blots shown 10 fold more protein extract was loaded in each Sec14^G266D lane compared to extracts containing wild type Sec14 for blots versus the T7 epitope due to protein expression level differences.</p

<i>sec14^ts</i> suppressing gene deletions.

<p><i>sec14^ts</i> suppressing gene deletions.</p

Membranes accumulate in growing cells expressing Sec14^G266D.

<p>Wild type cells and <i>sec14^ts</i> cells containing empty vector, a vector expressing wild type Sec14 on a low copy plasmid, or Sec14^G266D on a high copy (2 μ) plasmid, were grown at 25°C to mid-logarithmic phase and an aliquot transferred to 37°C for 1 hr followed by incubation in 1.5% KMnO₄, 1% sodium periodate, and then 1% NH₄Cl subsequent to embedding and viewing by transmission electron microscopy.</p

The levels of Sec14^G266D is regulated by the proteosome.

<p><i>A</i>, the level of Sec14 and Sec14^G266D in <i>sec14^ts rpn4</i>Δ cells. <i>B</i>, the level of Sec14 and Sec14^G266D in <i>sec14^ts</i> cells treated with MG132. Strains were transformed with plasmids expressing Sec14 or Sec14^G266D containing an N-terminal T7 epitope tag and were grown to mid-logarithmic phase at 25°C, with a subset shifted to 37°C for 2 hours (A). For MG132 treatment cells were grown as before and shifted to 37°C in the presence of 100 μM MG132 for 2 hours. Cells were disrupted by three passes through a French press and unbroken cells removed by centrifugation. Protein extract was separated by SDS-PAGE, transferred to PVDF membrane, and western blots versus the T7 epitope were performed. Pgk1 was used as load control.</p

Fus-Mid-GFP and

<p><b>Pma1 localization is defective in </b><b><i>sec14^ts</i></b><b> cells and restored by expression of Sec14^G266D. </b><i>A, sec14^ts</i> cells expressing Fus-Mid-GFP and also containing empty vector, a vector expressing wild type Sec14 on a low copy plasmid, or Sec14^G266D on a high copy (2 μ) plasmid, were grown at 25°C in 1% raffinose containing medium to mid-logarithmic phase. Cells were shifted to 37°C in pre-warmed 2% galactose containing medium for 3 hours. <i>B</i>, cells from <i>A</i> were quantified based on having only plasma membrane (PM) localization, only internal localization or both (vector n = 153, Sec14 n = 73, Sec14^G266D n = 107) <i>C</i>, the wild type <i>SEC14</i> gene was replaced with the <i>sec14^ts</i> allele in a yeast strain expressing chimeric Pma1-RFP. The strain was transformed with either empty vector, a plasmid carried at low copy (ARS/CEN) containing wild type Sec14, and low and high copy (2 μ) plasmids containing Sec14^G266D. Cells were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 16 hrs subsequent to determination Pma1-RFP localization by fluorescence microscopy.</p

Known vesicular trafficking pathways are still aberrant in growing cells expressing Sec14^G266D.

<p>The <i>sec14^ts</i> strain transformed with either empty vector, a plasmid carried at low copy (ARS/CEN) containing wild type Sec14, or a high copy (2 μ) plasmid containing Sec14^G266D were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 1 hr subsequent to determination of: <i>A</i>, invertase secretion (mean ± SE of three separate experiments performed in duplicate), <i>B</i>, or internal retention of Bgl2 at 2 and 16 hrs, similar results were seen at both time point with the 2 hr time point shown. <i>C</i>, the <i>sec14^ts</i> strain containing plasmid borne GFP-Snc1 was transformed with either empty vector, a plasmid carried at low copy (<i>ARS/CEN</i>) containing wild type Sec14, or a high copy (2 μ) plasmid containing Sec14^G266D. Cells were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 2 hrs. The localization of GFP-Snc1 was determined by fluorescence microscopy in live cells. <i>D</i>, The strains were grown at 25°C to mid-logarithmic phase and then transferred to 37°C for 15 min prior to the addition of FM4-64. The trafficking of FM4-64 in live cells was visualized by fluorescence microscopy.</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

Yeast strains used in this study.

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