Yeast Transformation and Growth Yeast cells were transformed by the lithium acetate method [41]. Transformants were incubated in liquid SG medium for 4 h and then diluted with water to OD600 0.05 and 0.005. Drops (5 mL) were spotted on plates and incubated at 20uC for 6–8 days (cold) or at 30uC for 2–3 days. All experiments were repeated independently at least three times. For isolation of total membrane protein fractions for immunodetection, 30–40 yeast colonies transformed with the desired plasmid(s) were inoculated in 100 mL of liquid SG medium in 250 mL flasks and grown at 30uC with 140 rpm shaking. After 24 hurs growth, cells were inoculated in 1 L of fresh SG medium in 2 L flasks and allowed to grow under the same conditions for a further 24 hours. Yeast Membrane Preparation and Protein Immunodetection Isolation of total cellular yeast membranes for protein expression analysis was performed as previously described [40]. Discontinuous sucrose gradient fractionation of plasma mem- brane-enriched membrane fractions was carried out essentially as described in [25] except that cells were grown for 24 hours (28oC, 160 rpm) in 50 mL selective SG media and then inoculated without washing into 500 mL fresh selective SG media before incubation for further 16 hoursF under the same conditions prior to membrane preparation. Protein samples were quantified by the method of Bradford using bovine serum albumin (BSA) as a standard. For protein blot analysis of membrane fractions, 30 mg total protein were precipitated with trichloroacetic acid (TCA), loaded onto SDS-PAGE gels, transferred to nitrocellulose membranes and immunodetected as previously described [26]. Detection of HA- and histidine (RGSH6 or RGSH10)-tagged proteins was carried out using a monoclonal anti-HA antibody (Sigma) and an anti-RGSHis antibody BSA-free (Qiagen), respectively. Dpm1p was immunodetected using an anti-dolichol phosphate mannose synthase antibody (Molecular Probes), Sed5p with affinity-purified anti-Sed5p [42] and Pma1p with a polyclonal antibody raised against its C terminus [43]. Bands were visualized with the 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazo- lium (BCIP/NBT) color development substrate (Promega). Transient Expression in Tobacco Epidermal Leaf Cells Agrobacterium tumefaciens strain C58C1 [44] was transformed by electroporation and transformants selected on YEP plates (1% yeast extract, 2% peptone, 1.5% agar) containing 25 mg/mL gentamycin and 50 mg/mL kanamycin. Transient expression in tobacco epidermal cells was carried out as described [25] using three week old N. benthamiana plants. Expression was visualized 4–5 days after infiltration. Plasmolysis of epidermal tobacco cells was achieved by infiltrating the leaves with a 1 M mannitol solution 1– 2 min prior to sample preparation for microscopic visualization. Confocal Microscopy A Leica TCS SP2/MP or SP5 II spectral confocal laser scanning microscope (Leica Microsystems, Heidelberg, Germany) with a 63x/1.2 numerical aperture water immersion objective were used as previously described [26]. Both GFP and YFP were excited at 488 nm and emission spectra were recorded between 495 and 510 nm for GFP (green channel) and 530 and 545 nm for YFP (magenta channel). Sequential scanning between lines was used to follow both fluorescent proteins at once. Autofluorescence was visualized between 600 and 680 nm. No fluorescence bleed- through of YFP or GFP signals was detected under our experimental conditions (see Figure S1). Plant Plasma Membrane P4 ATPase Supporting Information Figure S1 Bleed-through control of cells expressing GFP- and YFP-tagged proteins. A GFP::ALA1 fusion was transiently expressed in tobacco epidermal cells in the presence of an YFP tagged ALIS3. The image shows plasmolyzed cells subjected to osmotic shock in the presence of a concentrated mannitol solution. (A) Green: GFP fluorescence; (B) magenta: YFP fluorescence; (C) gray: bright field image. (D) overlay image. Two adjacent cells containing only GFP or only YFP signals are shown. In the upper part of the image, a cell containing both signals can be seen. Scale bar: 25 mm. (TIF) Figure S2 Alignment showing a comparison between ALA1 cDNA and the modified version used in for increasing expression of the protein in yeast. The unmodified ALA1 cDNA is highlighted in yellow. Silent mutations are highlighted in red (modification of 5 codons codifying for arginine and deletion of restriction sites) or magenta (elimination of a putative transcription termination signal). (TIF) Figure S3 ALA1 fails to complement the phenotypes of a yeast Ddrs2Ddnf1Ddnf2 mutant strain. A version of the ALA1 cDNA modified to eliminate a putative transcription termination site and five rare codons codifying for Arg (yeALA1) was expressed alone or together with an ALIS in yeast lacking the three endogenous P4 ATPases Drs2p, Dnf1p and Dnf2p. yeALA1 was expressed untagged or was tagged at the N-terminal end with an RGSH10-tag while ALIS proteins bear a RGSH6-tag at the same end. Left to right: glucose: control showing uninduced cells; galactose: control plate grown under standard conditions; 20uC: cells grown on galactose at the restrictive growth temperature; duramycin: cells grown on galactose plates containing 1.5 mM of the phosphatidylethanolamine-binding cytotoxic peptide duramy- cin; Pap. B: cells grown on galactose plates containing 0.05 mL/mL of the phosphatidylserine-binding cytotoxic peptide papuamide B; miltefosine: cells grown on galactose plates containing 2.5 mL/mL of the cytotoxic choline ether lipid miltefosine. (TIF) Figure S4 ALA1 reaches the plasma membrane when expressed in yeast. PM-enriched membranes from yeast expressing RGSH10:ALA1 alone or RGSH10:ALA1 together with RGSH6:ALIS1 were subjected to discontinuous sucrose density gradient fractionation. Fractions corresponding to 30 and 48% sucrose, enriched respectively in endomembranes (ER, Golgi) and plasma membranes, were analyzed. Western blots were probed using the following antibodies: anti-Pma1p, plasma membrane; anti-Sed5p, Golgi apparatus; anti-Dpm1p, ER; and anti-RGSHis, ALA1 and ALIS1. (TIF) Table S1 PCR Primers used in this work. Standard PCR was used to amplify the ALA1 genomic and cDNA fragments and the ALA1 promoter region. Overlapping PCR strategies were designed to modify the ALA1 cDNA fragment and the plasmid used for overexpression in yeast, as well as to generate a fusion of the ALA1 genomic fragment to its natural promoter for in planta expression. See Materials and Methods for further details. Artificial restriction sites before the ATG start codon or after the stop codon are marked in bold; Epitope tags are mark in bold and italics; New restriction sites generated by silent mutation within the codifying sequence are written in italics; Restriction sites (in brackets) and transcription termination sites eliminated by silent mutations are written in italic lowercase letters; Silent mutations PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e33042