Suppression of the growth defects in flippase mutants by decreased synthesis of PI.

<p>(A) Effects of inositol depletion on steady state phospholipid composition in the flippase mutants. Cells were grown in SD medium with or without inositol at 30°C for 12 h or 16 h (Neo1p-depleted). Phospholipids were extracted and quantified by liquid chromatography mass spectrometry as described in the Materials and Methods. The data represent mole percentage (mol%) of total phospholipids, with mean values ± standard deviation (n = 3). Asterisks indicate a significant difference in the Student’s <i>t</i> test (*: <i>P</i> < 0.05; **: <i>P</i> < 0.005). The strains used were YKT1066 (wild type, WT), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>). (B) Suppression of the cold-sensitive growth defects in the <i>cdc50</i>Δ mutant by partial depletion of Pis1p. Cells were pregrown to early log phase in SD medium containing 2% raffinose and 2% or 0.005% galactose for 2 days, washed, and adjusted to a concentration of 1.0 × 10⁷ cells/ml. Drops of 10 μl from 5-fold serial dilutions were spotted onto the same medium and YPDA, followed by incubation at the indicated temperature for the indicated time. The strains used were YKT1066 (wild type, WT), YKT1697 (<i>cdc50</i>Δ), YKT1942 (<i>P</i>_<i>GAL1</i><i>-PIS1</i>), and YKT1943 (<i>P</i>_<i>GAL1</i><i>-PIS1 cdc50</i>Δ). (C) Effects of Pis1p depletion on steady state phospholipid composition in the <i>cdc50</i>Δ mutant. Cells were pregrown and grown in SD medium containing 2% raffinose and 0.005% galactose for 4 days, in total, at 30°C. Phospholipids were extracted and quantified as in (A). The strains used were those in (B). (D) Aureobasidin A (AbA) treatment does not suppress the growth defects of flippase mutants. Cell growth was examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120108#pone.0120108.g001" target="_blank">Fig. 1A</a>. Cells were spotted onto a YPGA, YPDA, or YPDA containing 75 ng/ml AbA agar plate, followed by incubation at 30°C for 1 day. The strains used were YKT1066 (wild type, WT), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>). (E) <i>ART5</i> overexpression suppressed the growth defects of flippase mutants more efficiently than the <i>CHO1</i> overexpression. Cells were pregrown to early log phase in SG-Leu medium, washed, and adjusted to a concentration of 2.5 × 10⁷ cells/ml. Drops of 10 μl from 5-fold serial dilutions were spotted onto an SDA agar plate with or without inositol, followed by incubation at 30°C for 1 day or 1.5 days (the <i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ mutant). The strains used were YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), carrying YEplac181 (vector), pKT1259 (p<i>CDC50</i>), pKT1788 (p<i>NEO1</i>), pKT1719 (p<i>ART5</i>), or pKT1754 (p<i>CHO1</i>).