Ist2 Mediates ER-Plasma Membrane Contacts cytosolic face of the PM [5,6]. Therefore, we hypothesized that Ist2 recruits ER to the PM. The lipid-binding domain is positioned at the C-terminal end of a ,350 amino acid residues long cytosolic domain, providing enough flexibility to bridge the typical distance of 15 to 50 nm between cortical ER and PM [4]. To test our hypothesis, we performed immuno-EM with chemically fixed cells and detected the localization of Ist2. In all cells we observed perinuclear ER structures surrounding the nucleus (Fig. 2, labeled as N). In addition, we saw ER tubules located more to the centre of the cell (ER) and cortical ER (cER), which is positioned close to the PM and the cell wall (CW). Immuno-staining of WT cells with 10 nm gold-labeled Ist2-specific antibodies occasionally led to detection of Ist2 at sites where ER and PM were opposed (Fig. 2B, inset). In pTEF1-IST2 cells, we observed a strong accumulation of gold particles at the cortical ER and the PM (Fig. 2C, inset), suggesting that Ist2 localizes to cortical ER associated with the PM. The deletion of IST2 led to an increased distance between ER and PM (Fig. 2A, D). To quantify the amount of cortical ER in the three different strains, we defined ER membranes in proximity to the PM within distances of either 30, 100 or 200 nm. For each cell, we measured the total PM length in a section as well as the length of PM with underlying cortical ER within the defined threshold as schemat- ically illustrated in Fig. 2G. Finally, we calculated the percentage of PM that associates with cortical ER in each cell. This quantification revealed that in WT cells 48% of the PM is covered with cortical ER within 30 nm distance from the PM. Overexpression of Ist2 led to increased association of ER with the PM such as 91% of the PM had an underlying cortical ER (Fig. 2H). Indeed, the cortical ER covered most parts of the PM except invaginations (Fig. 2C, inset). In both cases theses values did not differ after quantifying ER membranes Figure 2. Ist2 recruits yeast ER to the PM. (A–F) Yeast cells expressing different amounts of Ist2 (ist2D in A and D, WT in B and E, and pTEF1-IST2 in C and F) were grown to log phase in YPD medium. (A–C) Cells were chemically fixed and ultrathin cryo-sections of gelatine-embedded cells were subjected to immuno-gold labelling using anti-Ist2 antibodies and protein A-10 nm gold. Lower panels show higher magnification of the indicated areas. Please note that cross sections in A–C are shown at different magnification. The average size of ist2D, WT and pTEF1-IST2 cells is similar, as revealed by light microscopy (data not shown). (D–F) Cells were high pressure frozen, freeze substituted and Epon embedded. Ribosome-studded ER is indicated by black arrowheads. (D) Ribosomes on the PM face of the cortical ER in ist2D are indicated by white arrowheads. Organelles and membranes are labelled (PM, plasma membrane; CW, cell wall; ER, endoplasmic reticulum; cER, cortical ER; N, nucleus; LD, lipid droplet). White bars correspond to 500 nm and black bars to 200 nm. (G) Cartoon illustrating the morphology of ER. For quantification PM and ER were traced in electron micrographs of representative cells after chemical fixation. The relative amount of PM associated with cortical ER within a defined distance (dotted line) was analyzed. (H) Quantification of PM fraction associated with cortical ER within a distance of 30 nm (white), 100 nm (grey), and 200 nm (black) in the indicated strains. Values represent the mean of independent images (ist2D: n = 12; WT: n = 10; pTEF1-IST2: n = 9). Error bars indicate the s.d. doi:10.1371/journal.pone.0039703.g002 PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e39703