Table 1.
S. cerevisiae strains used in this study.
Table 2.
Plasmids used in this study.
Fig 1.
Identification of ART5 as a multicopy suppressor of flippase mutations.
(A) Suppression of growth defects by overexpressing ART5 in Cdc50p-depleted mutants carrying a mutation synthetically lethal with cdc50Δ. Cells were grown to early log phase in SGA-Ura medium, washed, and adjusted to a concentration of 2.5 × 107 cells/ml. Drops of 4 μl from 5-fold serial dilutions were spotted onto a YPDA (Cdc50p-depleted) or YPGA (Cdc50p-expressed) agar plate, followed by incubation at 30°C for 1 day. The strains used were YKT1286 (PGAL1-CDC50 gcs1Δ), KKT116 (PGAL1-CDC50 fpk1Δ), and YKT1649 (PGAL1-CDC50 neo1–101), all carrying YEplac195 (vector), pKT1263 (pCDC50), or pKT1720 (pART5). (B) Suppression of growth defects in flippase mutants by overexpression of ART5. Cells were grown and examined as in (A), except that the PGAL1-CDC50 crf1Δ mutant was incubated at 25°C for 2 days. The strains used were YKT1511 (PGAL1-CDC50 crf1Δ), YKT1529 (PGAL1-CDC50 dnf1Δ crf1Δ), YKT1513 (PGAL1-CDC50 lem3Δ crf1Δ), and YKT1932 (PGAL1-NEO1), all carrying each of the plasmids in (A), except YKT1932 carried pKT1469 (pNEO1) as a positive control. (C) Suppression of the defects in membrane trafficking in flippase mutants by overexpression of ART5. Cells were grown in YPDA medium at 25°C for 14 h (Cdc50p-depleted crf1Δ) or at 30°C for 12 h (Neo1p-depleted), followed by microscopic observation of small- or middle-budded cells. The percent of cells with polarized GFP- or mRFP-Snc1p was determined (n>100) and is shown with the mean ± standard deviation of three independent experiments. Representative images are shown. The strains used were YKT1933 (PGAL1-CDC50 crf1Δ GFP-SNC1) and YKT1910 (PGAL1-NEO1 mRFP-SNC1), both carrying each of the plasmids in (A), except YKT1910 carried pKT1469 (pNEO1) as a positive control. Bar: 5 μm. (D) Failure of ART5 overexpression to suppress the alkylphosphocholine resistance and phospholipid-binding peptide sensitivity in a flippase mutant. Cells were grown to early log phase in SDA-Ura medium, washed, and adjusted to a concentration of 5.0 × 106 cells/ml. Drops of 10 μl and 4 μl from 5-fold serial dilutions were spotted onto SDA-Ura containing 5.0 μg/ml miltefosine and YPDA containing 0.5 μg/ml papuamide B (pap B) or 2.0 μM duramycin agar plates, respectively, followed by incubation at 30°C for 1 day. The strains used were YKT1066 (WT) and YKT715 (lem3Δ), both carrying YEplac195 (vector) or pKT1720 (pART5).
Fig 2.
The growth defects of flippase mutants were suppressed by mutations in inositol transporters.
(A) Suppression of the flippase mutations by overexpression was specific to ART5. Cell growth was examined as in Fig. 1A, except that the PGAL1-CDC50 crf1Δ mutant was incubated at 25°C for 2 days. The strains used were YKT1511 (PGAL1-CDC50 crf1Δ) and YKT1932 (PGAL1-NEO1), both carrying YEplac195 (vector), pKT1720 (pART5), pKT2135 (pRIM8), pKT2136 (pROD1), pKT2137 (pROG3), pKT1881 (pALY1), pKT1882 (pALY2), pKT2088 (pART5-PYm), or pKT2138 (pART5-AMm). YKT1511 and YKT1932 also carried pKT1263 (pCDC50) and pKT1469 (pNEO1), respectively, as positive controls. (B) Suppression of the growth defects of flippase mutants by mutations in inositol transporters. Cells were pregrown to early log phase in SGA-Ura or YPGA medium, and cell growth was examined as in Fig. 1A. The strains used were YKT1066 (wild type, WT), YKT1866 (itr1Δ itr2Δ), YKT1529 (PGAL1-CDC50 dnf1Δ crf1Δ), YKT1934 (PGAL1-CDC50 dnf1Δ crf1Δ itr1Δ), YKT1935 (PGAL1-CDC50 dnf1Δ crf1Δ itr1Δ itr2Δ), YKT1513 (PGAL1-CDC50 lem3Δ crf1Δ), YKT1915 (PGAL1-CDC50 lem3Δ crf1Δ itr1Δ), YKT1914 (PGAL1-CDC50 lem3Δ crf1Δ itr1Δ itr2Δ), YKT1932 (PGAL1-NEO1), YKT1877 (PGAL1-NEO1 itr1Δ), and YKT1881 (PGAL1-NEO1 itr1Δ itr2Δ). YKT1529, YKT1513, and YKT1932 also carried YEplac195 (vector) or pKT1720 (pART5).
Fig 3.
Suppression of the defects in flippase mutants by depletion of inositol from culture medium.
(A) Suppression of the growth defects. Cells were pregrown to early log phase in YPGA 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 SD agar plate with or without inositol, followed by incubation at 30°C for 1.5 days. The strains used were YKT1066 (wild type, WT), YKT1529 (PGAL1-CDC50 dnf1Δ crf1Δ), YKT1513 (PGAL1-CDC50 lem3Δ crf1Δ), YKT1932 (PGAL1-NEO1), YKT1909 (PGAL1-NEO1 PGAL1-CDC50), and YKT1887 (PGAL1-NEO1 PGAL1-CDC50 lem3Δ crf1Δ). (B) Suppression of growth defects in the flippase null mutants. Cells were pregrown to early log phase in SD medium without inositol, and cell growth was examined as in (A), except that they were incubated at 30°C, 25°C, or 18°C for 1.5, 2, or 5 days, respectively. The strains used were YKT1066 (wild type, WT), YKT1944 (cdc50Δ), and YKT1945 (cdc50Δ dnf1Δ). YKT1945 was constructed by tetrad dissection of spores from a cdc50Δ/CDC50 DNF1/dnf1Δ heterozygous diploid on an inositol-depleted SD agar plate. (C) Suppression of the defects in endocytic recycling of Snc1p. Cells were grown in SD medium with or without inositol at 30°C for 12 h or 16 h (Neo1p-depleted), followed by microscopic observation of small- or middle-budded cells. The percent of cells with mRFP-Snc1p in polarized sites was determined (n>100) and is shown with the mean ± standard deviation of three independent experiments. Representative images are shown. The strains used were YKT1912 (mRFP-SNC1, WT), YKT1936 (PGAL1-CDC50 dnf1Δ crf1Δ mRFP-SNC1), YKT1937 (PGAL1-CDC50 lem3Δ crf1Δ mRFP-SNC1), and YKT1910 (PGAL1-NEO1 mRFP-SNC1). Bar: 5 μm. (D) Restoration of the plasma membrane location of GFP-Snc1p-pm. Cells were grown in SD-Ura or SD-Leu medium with or without inositol at 30°C for 16 h. More than 100 cells were microscopically observed, and the percent of cells with internally accumulated GFP-Snc1p-pm was determined. The strains used were YKT1066 (wild type, WT), YKT1120 (PGAL1-CDC50 lem3Δ crf1Δ), and YKT1660 (PGAL1-NEO1), carrying pKT1444 (pRS416-GFP-SNC1-pm) or pKT1491 (pRS315-GFP-SNC1-pm). Bar: 5 μm. (E) Formation of secretory vesicles. Cells were grown in SD with or without inositol at 30°C for 12 h, followed by a shift to 37°C for 2 h. Secretory vesicles were fractionated by the Nycodenz density gradient, and each fraction was measured for mRFP fluorescence intensity and the total amount of phospholipid phosphates. Strains used were YKT1844 (sec6–4 mRFP1-Lact-C2) and YKT1854 (sec6–4 PGAL1-CDC50 lem3Δ crf1Δ mRFP1-Lact-C2).
Fig 4.
Flippase mutations are not suppressed by activation of either the Ino2p-Ino4p pathway or the unfolded protein response.
(A) Activation of the Ino2p-Ino4p pathway by the opi1Δ mutation does not suppress the growth defects of flippase mutants. Cell growth was examined as in Fig. 1A, 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 (opi1Δ), YKT1510 (PGAL1-CDC50 crf1Δ), YKT1939 (PGAL1-CDC50 crf1Δ opi1Δ), YKT1120 (PGAL1-CDC50 lem3Δ crf1Δ), YKT1940 (PGAL1-CDC50 lem3Δ crf1Δ opi1Δ), YKT1660 (PGAL1-NEO1), and YKT1941 (PGAL1-NEO1 opi1Δ). (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 HAC1 (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 (hac1Δ), YKT1529 (PGAL1-CDC50 dnf1Δ crf1Δ), YKT1513 (PGAL1-CDC50 lem3Δ crf1Δ), and YKT1932 (PGAL1-NEO1), all carrying either YCplac111 (Vector) or pKT2139 [pHAC1 (238 type S238A)]. (C) Treatment with TM or DTT. Cell growth was examined as in Fig. 1A. 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 (hac1Δ), YKT1529 (PGAL1-CDC50 dnf1Δ crf1Δ), YKT1513 (PGAL1-CDC50 lem3Δ crf1Δ), and YKT1932 (PGAL1-NEO1).
Fig 5.
Suppression of the growth defects in flippase mutants by decreased synthesis of PI.
(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 t test (*: P < 0.05; **: P < 0.005). The strains used were YKT1066 (wild type, WT), YKT1529 (PGAL1-CDC50 dnf1Δ crf1Δ), YKT1513 (PGAL1-CDC50 lem3Δ crf1Δ), and YKT1932 (PGAL1-NEO1). (B) Suppression of the cold-sensitive growth defects in the cdc50Δ 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 (cdc50Δ), YKT1942 (PGAL1-PIS1), and YKT1943 (PGAL1-PIS1 cdc50Δ). (C) Effects of Pis1p depletion on steady state phospholipid composition in the cdc50Δ 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 Fig. 1A. 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 (PGAL1-CDC50 dnf1Δ crf1Δ), YKT1513 (PGAL1-CDC50 lem3Δ crf1Δ), and YKT1932 (PGAL1-NEO1). (E) ART5 overexpression suppressed the growth defects of flippase mutants more efficiently than the CHO1 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 PGAL1-CDC50 lem3Δ crf1Δ mutant). The strains used were YKT1529 (PGAL1-CDC50 dnf1Δ crf1Δ), YKT1513 (PGAL1-CDC50 lem3Δ crf1Δ), and YKT1932 (PGAL1-NEO1), carrying YEplac181 (vector), pKT1259 (pCDC50), pKT1788 (pNEO1), pKT1719 (pART5), or pKT1754 (pCHO1).