Ist2 Mediates ER-Plasma Membrane Contacts Figure 3. Overexpression of Ist2 traps the perinuclear ER at the PM. (A–C) Electron micrographs of log phase pTEF1-IST2 cells. (A and B) Cells were high pressure frozen, freeze substituted and Epon embedded. (B) Arrowheads indicate the association of cortical ER with the PM. (C) Immuno- gold labeling of chemically fixed cells using anti-Ist2 antibodies. Organelles and membranes are labeled (PM, plasma membrane; CW, cell wall; ER, endoplasmic reticulum; N, nucleus; NP, nuclear pore). The white bar corresponds to 1 mm and the black bar to 200 nm. (D) Association of nuclei analyzed by CLS microscopy of ist2D, WT, and pTEF1-IST2 cells expressing GFP-HDEL (n = 39, 38, 55), Sec63-GFP (n = 46, 60, 48), GFP-Ubc6 (n = 66, 54, 50), and GFP-Scs2 (n = 52, 74, 95). Class 1 cells have the nucleus in the centre without any contact to the periphery (white), class 2 cells have the nucleus in contact with the periphery (grey), and class 3 cells have a deformed nucleus that aligns with the periphery (black). Quantification was performed with blinded data. doi:10.1371/journal.pone.0039703.g003 Pearson’s coefficient for colocalization ranged from 0.61660.075 to 0.72860.069 (Fig. S4B). Colocalization of dsRed-HDEL with Sec63-GFP and GFP-Ubc6 revealed a better overlap (Pearson’s coefficient of 0.94960.035 and 0.9560.037; Fig. S4B, E and G), suggesting a partial segregation of Ist2 into subdomains of the cortical ER. However, the segregation of membrane proteins in the cortical ER was not specific for Ist2. The colocalization of Yop1-mCherry with GFP-HDEL was similar to mCherrry-Ist2 with GFP-HDEL (Pearson’s coefficient of 0.78660.07 and 0.61660.075; Fig. S4B, C and D). Colocalization of Sec63-GFP and GFP-Ubc6 with Yop1-mCherry showed Pearson’s coefficients of 0.71660.125 and 0.79760.084 (Fig. S4B, F and H). Association of Ist2 with domains of the PM The impaired adaptation of ist2D cells, when diluted from cultures with an OD600.2.0 (Fig. 1A–C) may result from a Figure 4. Ist2 colocalizes with proteins of the cortical ER. Surface views of epifluorescence microscopy with ist2D cells expressing mCherry- Ist2. These cells co-express GFP-HDEL, Sec63-GFP, GFP-Ubc6 from CEN plasmids or Yop1-GFP. The merged images show mCherry-Ist2 in magenta and the respective ER proteins in green. The fourth panels show intensity profiles of the indicated cross sections in the merged images. doi:10.1371/journal.pone.0039703.g004 PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e39703