Cell surface PE is reduced in cells carrying the pdr5Δ mutation. KCY1112 (wild-type), KCY689 (pdr5Δ), KCY1011 (yor1Δ pdr5Δ), and KCY1141 (lem3Δ) cells were diluted to 0.05 OD₆₀₀ unit/ml in YPD medium containing 50 μM Ro (A) or 200 μM MG132 (B). After a 9-h incubation, the OD₆₀₀ of each culture was measured. Values indicate the OD₆₀₀ for each cell line in the presence of the drugs, relative to that in the absence of the drugs. Values represent the means ± SD from three independent experiments. The statistical significance of each difference as compared with results from wild-type cells was determined using a two-tailed Student's t test. *p < 0.05; **p < 0.01.

MCK1, MOT3, RIM13, RIM20, and RIM21 are involved in Rsb1 expression induced by changes in lipid asymmetry. KCY662 (wild-type), KCY689 (pdr5Δ), KCY1012 (mck1Δ pdr5Δ), KCY1013 (mot3Δ pdr5Δ), KCY694 (pdr1Δ pdr5Δ), KCY1014 (rim13Δ pdr5Δ), KCY697 (rim20Δ pdr5Δ), and KCY1015 (rim21Δ pdr5Δ) cells were grown in YPD medium. (A and B) Total proteins were prepared from each culture. (A) Proteins (13 μg) were separated by SDS-PAGE and then subjected to immunoblotting with an anti-HA antibody or, to demonstrate uniform protein loading, an anti-Pgk1 antibody. (B) Proteins (1.7 μg) were treated with Endo H, separated by SDS-PAGE, and subjected to immunoblotting as in A. (C) Total cell lysates were prepared from each culture and subjected to β-galactosidase reporter assays. Values indicate the means ±SD from three independent experiments. The statistical significance of each difference as compared with results from pdr5Δ cells was determined using a two-tailed Student's t test. *p < 0.01.

The pH-responsive Rim101 pathway is involved in lipid asymmetry signaling. (A and B) Cells carrying single or double mutations were grown in YPD medium. Total proteins (1.7 μg) prepared from each culture were incubated with Endo H, separated by SDS-PAGE, and subjected to immunoblotting with an anti-HA or anti-Pgk1 antibody. (A) KCY689 (pdr5Δ), KCY1014 (rim13Δ pdr5Δ), KCY697 (rim20Δ pdr5Δ), KCY1015 (rim21Δ pdr5Δ), and KCY1016 (rim101Δ pdr5Δ) cells were used. (B) KCY662 (wild-type), KCY1103 (mot3Δ), KCY1102 (mck1Δ), KCY1104 (rim101Δ), KCY692 (lem3Δ), KCY1063 (mot3Δ lem3Δ), KCY1062 (mck1Δ lem3Δ), and KCY1064 (rim101Δ lem3Δ) cells were used. (C and D) KCY689 (pdr5Δ), KCY1015 (rim21Δ pdr5Δ), KCY1046 (dfg16Δ pdr5Δ), KCY1047 (rim9Δ pdr5Δ), KCY1048 (rim8Δ pdr5Δ), KCY1014 (rim13Δ pdr5Δ), KCY697 (rim20Δ pdr5Δ), KCY1049 (ygr122wΔ pdr5Δ), KCY1051 (vps28Δ pdr5Δ), KCY1052 (vps25Δ pdr5Δ), KCY1053 (snf7Δ pdr5Δ), KCY1054 (vps20Δ pdr5Δ), KCY1055 (did4Δ pdr5Δ), and KCY1056 (vps24Δ pdr5Δ) cells bearing the pFI1 (HA-RIM101) or pIKD493 (P_RSB1-lacZ) plasmid were used. (C) Cells harboring the plasmid pFI1 were precultured in SC medium lacking leucine, transferred to YPD medium, and grown to logarithmic phase. Total proteins were prepared from each culture and incubated with Endo H. Proteins (1.25 μg for Rsb1-HA and Pgk1 blots, and 1.7 μg for HA-Rim101 blots) were separated by SDS-PAGE and subject to immunoblotting with an anti-HA or anti-Pgk1 antibody. (D) Cells harboring the plasmid pIKD493 were precultured in SC medium lacking histidine, transferred to YPD medium, and grown to logarithmic phase. Total cell lysates were prepared from each culture and subjected to β-galactosidase reporter assays. Values represent the means ± SD from three independent experiments. The statistical significance of each difference as compared with results from the pdr5Δ cells bearing pIKD493 was determined using a two-tailed Student's t test. (* p < 0.05) (E) KCY662 (wild-type) and KCY1104 (rim101Δ) cells harboring the pRS315 or pIKD509 (Myc-RIM101-531) plasmid were cultured in SC medium lacking leucine and transferred to YPD medium. Total proteins prepared from each culture were incubated with Endo H. Proteins (2.5 μg for Myc-Rim101-531 blots and 1.25 μg for Rsb1-HA and Pgk1 blots) were separated by SDS-PAGE and subjected to immunoblotting with an anti-Myc, anti-HA, or anti-Pgk1 antibody. (F) KCY662 (wild-type), KCY689 (pdr5Δ), and KCY692 (lem3Δ) cells, each bearing the pFI1 plasmid, were cultured in SC medium lacking leucine for 3 h. An equal volume of buffered SC medium lacking leucine was added to the culture medium, and the cells were incubated for another 2 h. Total proteins were prepared from each culture and incubated with Endo H. Proteins (1.7 μg for HA-Rim101 blots, and 1.25 μg for Rsb1-HA and Pgk1 blots) were separated by SDS-PAGE and subject to immunoblotting with an anti-HA or anti-Pgk1 antibody.

The rim101Δ mutation and the mot3Δ or mck1Δ mutation exhibit additive effects in Rsb1 induction. KCY689 (pdr5Δ), KCY1013 (mot3Δ pdr5Δ), KCY1065 (mot3Δ mck1Δ pdr5Δ), KCY1012 (mck1Δ pdr5Δ), KCY1067 (mck1Δ rim101Δ pdr5Δ), KCY1016 (rim101Δ pdr5Δ), and KCY1066 (rim101Δ mot3Δ pdr5Δ) cells harboring the pIKD493 (P_RSB1-lacZ) plasmid were grown in SC medium lacking histidine. Total cell lysates were prepared from each culture and subjected to β-galactosidase reporter assays. Values represent the means ± SD from three independent experiments. The statistical significance of each difference indicated was determined using a two-tailed Student's t test. *p < 0.01.

The Rim101 pathway is activated by changes in lipid asymmetry. (A) Total proteins (1.25 μg) prepared from KCY662 (wild-type), KCY689 (pdr5Δ), KCY1011 (yor1Δ pdr5Δ), KCY692 (lem3Δ), KCY1029 (dnf1Δ dnf2Δ), and KCY696 (lem3Δ pdr5Δ) cells, each harboring the pFI1 (HA-RIM101) plasmid, were incubated with Endo H and separated by SDS-PAGE, followed by immunoblotting with an anti-HA or anti-Pgk1 antibody. (B) The intensities of the band for HA-Rim101 and HA-Rim101ΔC presented in A were quantified using Image J software (http://rsb.info.nih.gov/ij/) and are expressed as a percentage reflecting the level of HA-Rim101ΔC relative to sum of the level of the HA-Rim101 plus HA-Rim101ΔC. Values represent the means ± SD from the experiment shown in A and three other independent experiments. The statistical significance of each difference as compared with results from wild-type cells bearing pFI1 was determined using a two-tailed Student's t test. *p < 0.05.

RSB1 gene expression is repressed by Nrg1, downstream of Rim101. (A) KCY689 (pdr5Δ), KCY1016 (rim101Δ pdr5Δ), KCY1060 (ime1Δ pdr5Δ), KCY1061 (nrg1Δ pdr5Δ), and KCY1070 (rim101Δ nrg1Δ pdr5Δ) cells were grown in YPD medium. Total proteins (1.7 μg) prepared from each culture were incubated with Endo H, separated by SDS-PAGE, and subject to immunoblotting with an anti-HA or anti-Pgk1 antibody. (B) Schematic representation of the position and sequences of NREs. The locations of the 5′-nucleotides of NREs are at −761 (NRE1), −470 (NRE2), and −460 (NRE3) relative to the transcription start site. The sequences of the mt1-3 mutations are also shown. (C) The pRS423 (vector), pIKD412 (wild-type; wt), pIKD414 (mt1), pIKD416 (mt2), pIKD418 (mt3), and pIKD420 (mt2/3) plasmids were introduced into SEY6210 (wild-type), KCY595 (rim101Δ), and KCY594 (nrg1Δ) cells. Cells were precultured in SC medium lacking histidine, transferred to YPD medium, and grown to logarithmic phase. Total proteins (1.25 μg) prepared from each culture were treated with Endo H, separated by SDS-PAGE, and subject to immunoblotting with an anti-HA or anti-Pgk1 antibody.

Model of the lipid asymmetry signaling pathway. A signal from moderate increases in pH induces expression of the pH-responsive genes through activation of the Rim101 pathway. The same Rim101 pathway transduces a signal resulting in Rsb1 expression, but other pathways, such as that involving Mot3 and Mck1, are also required. Changes in lipid asymmetry, caused either by a flip or flop mutation, induce activation of the Rim101 pathway and probably of the Mot3/Mck1-related pathways as well. However, these pathways are activated differently by the flip or flop mutation. The Rim101 pathway is activated more strongly by the flip mutation, whereas other pathways seem to be activated more predominantly by the flop mutation. Pdr1 may act as a basic transcription factor, since little Rsb1 expression was observed in pdr1Δ cells.