Flippase mutations are not suppressed by activation of either the Ino2p-Ino4p pathway or the unfolded protein response.

<p>(A) Activation of the Ino2p-Ino4p pathway by the <i>opi1</i>Δ mutation 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>, except the cells were incubated at 30°C or 25°C for 1 day or 2 days, respectively. The strains used were YKT38 (wild type, WT), YKT1938 (<i>opi1</i>Δ), YKT1510 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ), YKT1939 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ <i>opi1</i>Δ), YKT1120 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), YKT1940 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ <i>opi1</i>Δ), YKT1660 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), and YKT1941 (<i>P</i>_<i>GAL1</i><i>-NEO1 opi1</i>Δ). (B, C) Activation of the unfolded protein response pathway (UPR) does not suppress the growth defects of flippase mutants. (B) Expression of the UPR-activating <i>HAC1</i> (238 type S238A) mutant. Cells were grown in SG-Leu medium, washed, and adjusted to a concentration of 1.0 × 10⁷ cells/ml. Drops of 10 μl from 5-fold serial dilutions were spotted onto an SG-Leu, SD-Leu, or SD-Leu without inositol agar plate, followed by incubation at 30°C for 1.5 days. The strains used were KKT466 (<i>hac1</i>Δ), 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>), all carrying either YCplac111 (Vector) or pKT2139 [p<i>HAC1</i> (238 type S238A)]. (C) Treatment with TM or DTT. 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 containing 0.5 μg/ml TM, or SD containing 6.5 mM DTT agar plate, followed by incubation at 30°C for 1 day. The strains used were YKT1066 (wild type, WT), KKT466 (<i>hac1</i>Δ), 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>).