Pearsons’ coefficient was determined using the JACoP plugin [57] in ImageJ. Transmission Electron Microscopy For immuno-EM yeast cells were grown to mid log phase in YPD medium at 30uC, concentrated by filtration and chemically fixed according to [58]. In detail, concentrated cells were mixed with double-concentrated fixative [4% (w/v) paraformaldehyde, 0.4% (v/v) glutaraldehyde in 0.1 M PHEM buffer (20 mM PIPES, 50 mM HEPES, pH 6.9, 20 mM EGTA, 4 mM MgCl2)] and incubated at room temperature for 30 min. Then, cells were pelleted, resuspended in standard fixative (2% (w/v) paraformal- dehyde, 0.2% (v/v) glutaraldehyde in 0.1 M PHEM buffer) and incubated at 4uC overnight. Fixed cells were washed 3 times in PHEM buffer and incubated in 1% (w/v) periodic acid in PHEM buffer for 1 hour at 4uC and again washed 3 times. Cells were then embedded in 12% (w/v) gelatine in PHEM buffer, cut into small blocks, infused with 2.3 M sucrose in 0.1 M PHEM buffer at 4uC overnight, mounted on metal pins and frozen in liquid nitrogen. Ultrathin cryo-sections (75 nm) were cut using a cryo-ultramicro- tome (Ultracut UCT with EM FCS, Leica) and a diamond knife (Diatome) at 2110uC and placed on formvar-coated nickel grids. For immuno-labeling, sections were incubated with polyclonal rabbit Ist2 anti-serum [59] for 20 min, followed by incubation with protein A-10 nm gold (CMC, Utrecht) for 20 min. Sections were contrasted with 0.4% (w/v) uranyl acetate in 2 M methyl- cellulose for 10 min on ice and embedded in the same solution. For Epon embedding, cells were grown as described above, rapidly filtered and vitrified using an EM HPM100 (Leica) high- pressure freezer. Fixed cells were further processed by freeze substitution in a Leica EM AFS, i.e. incubated in 0.5% glutaraldehyde, 1% H2O in acetone at 290uC for 70 hours, followed by incubation in 0.5% glutaraldehyde, 0.1% uranyl acetate, 5% H2O in acetone for 7 hours. Temperature was raised to 220uC (+5uC/hour), kept constant for 18 hours, and further raised to 4uC (+10uC/hour). Samples were washed 3 times with acetone, twice with propylene oxide and metal HPF planchets were removed. Samples were first infiltrated with 50% Epon resin in propylene oxide for 1 hour at room temperature, then with 100% Epon for an additional hour, and with fresh Epon resin overnight. After replacement with fresh resin, samples were incubated for 30 hours at 60uC. 70 nm ultrathin sections were cut with an ultramicrotome (Ultracut UCT, Leica) and contrasted with lead citrate. All sections were examined with a Philips CM120 transmission electron microscope and micrographs were acquired with a CCD camera (Megaview III, Olympus Soft Imaging Systems). Image processing was performed using iTEM software (Olympus Soft Imaging Systems). Generation of yeast membranes and Western blotting For fractionation, yeast cells were disrupted in lysis buffer (50 mM Hepes-KOH pH 7.6, 50 mM potassium acetate, 5 mM magnesium acetate, 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, Complete Protease Inhibitor (Roche)) and pelleted at 25000 g [59] [59]. Membrane fractions were resuspended in HU-buffer (8 M urea, 5% (w/v) SDS, 200 mM Tris–HCl, pH 6.8, 1 mM EDTA, 0.05% (w/v) bromphenol blue, 4% (v/v) b-mercaptoethanol), incubated for 10 min at 50uC and separated by 7.5% SDS–PAGE followed by Western blotting using Ist2- (1:25000), Sec61- (1:7500 [59,60]), GFP- (1:20,000, rabbit serum, gift from Dirk Go¨ rlich, Max-Planck-Institute for Biophysical Chemistry, Go¨ ttingen, Ger- many), Pma1 (1:10.000, rabbit serum, provided by Ramo´ n Serrano, Universidad Polite´cnica de Valencia, Valencia, Spain) Ist2 Mediates ER-Plasma Membrane Contacts or ANO1-specifc antibodies. The ANO1-specific antibodies were raised in a rabbit against KLKQQSPPDHEECVKRKQ- RYEVDY and CRYKDYREPPWSENK peptides. Detection and quantification of protein bands from Western blots was done using the LAS-1000 system (Fujifilm) followed by image processing using Adobe Photoshop and ImageJ software. pH measurements Cytosolic pH was measured using pH-sensitive GFP (pHluorin, [34]) expressed under the control of the TEF1 promoter from a CEN plasmid (pMS588). Fluorescence intensities at excitation wavelengths of 390 and 480 nm were measured at a constant emission wavelength of 520 nm in a FLUOstar Omega plate reader (BMG). Calibration of fluorescence with pH was carried out with 0.1% digitonin permeabilized cells in HC media buffered with 50 mM 3-(N-morpholino)propanesulfonic acid to 6.0, 6.5, 7.0, and 7.5. Supporting Information Figure S1 Log growth of pTEF1-IST2 is similar to WT. (A–B) ist2D cells were transformed with HIS3 or URA3 CEN plasmids encoding IST2 resulting in strains ist2D+IST2 (CEN- HIS3) and ist2D+IST2 (CEN-URA3). (A) Growth of ist2D+IST2 (CEN-HIS3) and ist2D+IST2 (CEN-URA3) relative to WT (set to 100%) after 10 hours. Cultures were diluted to OD600 0.05 from pre-cultures with an OD600 1.0–2.0 (log), OD600 2.0–2.5 (late log), OD600 2.5–3.0 (transition), and OD600.3.0 (stationary). Error bars depict s.d., non-significant differences are indicated as n.s. (B) Membranes prepared from 5 OD600 of WT cells transformed with an empty plasmid (pRS303), and ist2D+IST2 (CEN-HIS3), and ist2D+IST2 (CEN-URA3) were separated. Ist2 and Sec61 were detected with specific antibodies. (C) Stationary cultures (OD600.3.0) were diluted to 0.05 OD600 and grown at 25uC in HC complete medium for 30 hours. Growth curves are plotted with linear x-axis (time [h]) and logarithmic ordinate (OD600). (TIF) Figure S2 Overexpression of Ist2 led to an increase of cortical ER. (A) Quantification of the length of the PM with underlying cortical ER containing GFP-HDEL, Sec63-GFP or GFP-Ubc6. The ER proteins GFP-HDEL, Sec63-GFP, and GFP- Ubc6 were localized in ist2D, WT, and pTEF1-IST2 cells using epifluorescence microscopy. The intensities of the peripheral signals of equatorial sections were quantified. Areas with more than 70% of the average peripheral signal intensity were classified as PM with an underlying cortical ER. For comparison the quantification of cortical ER by EM from figure 2H is shown as black bars. (B) Examples of ist2D, WT, pTEF1-IST2 strains expressing GFP-Ubc6. Intensity profiles of peripheral staining as indicated by arrows in left panels are plotted in right panels. The percentage of PM with and without an underlying cortical ER is shown as cER and PM, respectively. (C) Representative z-stack CLS images of ist2D and pTEF1-IST2 cells. All cells express the PM-marker Pma1-mCherry (in magenta) and coexpress either GFP-HDEL, Sec63-GFP, GFP-Ubc6 or GFP-Scs2 (all shown in green). The scale bar corresponds to 2 mm. (TIF) Figure S3 Inheritance of cortical ER into the growing bud is independent of Ist2. (A) Quantification of fluorescence ratios between daughter and mother cells in ist2D (white), WT (black) and pTEF1-IST2 (grey) cells expressing both Yop1- mCherry and Sec63-GFP. Daughter cells were classified as small (S; ,0.4 daughter perimeter/mother perimeter), medium (M; 0.4– PLoS ONE | www.plosone.org 11 July 2012 | Volume 7 | Issue 7 | e39703