within 100 or 200 nm distance from the PM demonstrating that cortical ER associated within a distance of 30 nm with the PM. In ist2D cells the amount of PM-associated cortical ER is substantially reduced. Compared to 48% in WT cells only 8% of PM is covered with cortical ER when quantified within a distance of 30 nm (Fig. 2H). Quantifying ER membranes within increasing distances to the PM from 30 nm to 100 nm and 200 nm revealed that in ist2D some cortical ER can be found at larger distances from the PM. In comparison to WT and pTEF1-IST2 cells in which the amount of cortical ER stays constant within all defined distances, this increase demonstrates that cortical ER is less close associated with the PM in ist2D cells. As an independent method for the quantification of PM-associated ER, we employed fluorescence light microscopy and measured the length of the PM with cortical ER staining in cross sections. For this purpose, we expressed three types of GFP-tagged ER proteins: a soluble luminal protein (GFP-HDEL), an integral membrane protein (Sec63-GFP), and a tail-anchored protein (GFP-Ubc6). We found that overexpression of Ist2 in pTEF1-IST2 cells led to an increased amount of PM with associated cortical ER (Fig. S2A), in agreement with the EM data. In ist2D and WT cells, however, we observed no difference in the association of the cortical ER with the PM. This is consistent with previous results [6], showing that deletion of IST2 did not lead to a reduction of cortical ER staining within the resolution of standard light microscopy. This difference could be explained by the accumulation of peripheral ER at a distance to the PM that is not resolved by light microscopy, where the maximal resolution limit is 200 nm. Indeed, using 200 nm as a threshold to quantify cortical ER in our EM pictures revealed that 48.5% (69.7%) of the WT PM and as much as 29% (66%) of the ist2D PM is covered with cortical ER. The remaining discrepancy between values obtained by light microscopy and EM results either from the fact that the resolution limit in our light microscopy is larger than 200 nm or from the technical difference of using fixed cells for EM and living cells for light microscopy. In live-cell imaging by light microscopy additional fluorescence from extra-focal planes is recorded. This clearly demonstrates that the resolution achieved by EM is critical to determine the amount of Ist2-dependent cortical ER. To better preserve the ultra structure of the yeast cells and to analyze ER morphology, ist2D, WT and pTEF1-IST2 cells were vitrified using high pressure freezing, freeze substituted and Epon embedded. Analyses of membranes in such cells showed that the cortical ER is studded with ribosomes, which were seen as black dots (Fig. 2D–F, indicated by arrowheads). In WT and pTEF1-IST2 cells the ribosomes were restricted to the cytosolic face of the cortical ER (Fig. 2E, F). The space between ER and PM was free of ribosomes and appeared transparent. In ist2D this was seen at some parts of the cortical ER but most of the cortical ER was covered with ribosomes on both sides (Fig. 2D, indicated by white arrowheads). The loss of Ist2 led to an increased distance between ER and PM, which allows ribosomes with a diameter of approximately 25 nm to occupy the space between ER and PM. On the other hand the overexpression of Ist2 resulted in an increase of cortical ER tightly opposed to the PM. Taken together, these data support a function of Ist2 as a membrane tether or as a protein that enables tethering performed by other components. Ist2-mediated recruitment of ER to the PM may create a specific ribosome-free subcompartment of the cytosol positioned between two adjacent membranes. Increased ER-PM contact formation by overexpression of Ist2 traps the nucleus at the PM In cells overexpressing Ist2, we often observed an association of the nuclei with the cell periphery (Fig. 3A–C). The nuclear envelope was associated with the PM over a distance of more than Ist2 Mediates ER-Plasma Membrane Contacts 1 mm. This tight association of perinuclear ER and PM resulted in deformation of the nucleus. Cortical ER that emanated from the perinuclear ER became associated with the PM (Fig. 3B, indicated by arrowheads). Immuno-gold labeling with Ist2 antibodies showed staining at regions where the nuclear envelope aligns with the PM but not at other regions of the nuclear envelope. This suggests that Ist2 specifically localizes to ER domains that are in close proximity to the PM (Fig. 3C). In order to quantify this effect of Ist2 overexpression, we analyzed the nuclear morphology in ist2D, WT and pTEF1-IST2 cells expressing GFP-HDEL, Sec63-GFP, GFP-Ubc6, or GFP- Scs2 by confocal laser scanning (CLS) microscopy. Cells were divided in three classes with (1) the nucleus in the centre without any contact to the periphery, (2) the nucleus in contact with the periphery, and (3) a deformed nucleus in contact with the periphery (Fig. 3D). Examples for the localization of the individual ER proteins in all classes are shown in figure S2C. Overexpression of Ist2 caused the accumulation of deformed nuclei at the cell periphery in the majority of cells (class 3), and nuclei of almost all remaining cells also showed contacts to the periphery (class 2). In contrast, fewer contacts between the nuclei and the periphery were detected in WT and ist2D cells. Next, we asked whether Ist2 contributes to the inheritance of ER into daughter cells and coexpressed Sec63-GFP and Yop1- mCherry in ist2D, WT and pTEF1-IST2 cells. Sec63 is an integral membrane protein involved in posttranslational protein translo- cation. Yop1 is a member of the reticulon protein family and insertion of its hairpin-like hydrophobic region into the cytosolic face of ER induces membrane curvature [29]. The expression of Yop1-GFP was decreased in ist2D but the protein remained peripherally localized (Fig. S3E and F). Overexpression of Ist2 led to accumulation of Yop1-GFP in large cortical structures resembling cisternae but did not change the expression level. A comparison of the fluorescence ratios between daughter and mother cells revealed an accumulation of Yop1-mCherry in small daughter cells, whereas Sec63-GFP was equally distributed (Fig. S3A and B). This indicates a sequential inheritance of specific ER domains into daughter cells [4]. The deletion of IST2 had no effect, whereas overexpression of Ist2 had only a small effect on the inheritance of ER into small daughters (Fig. S3A). As described above, overexpression of Ist2 caused an accumulation of Sec63- containing perinuclear ER at the cell periphery (Fig. S3D). In summary, these data suggest that proper ER morphology and position of the nucleus are sensitive to the abundance of Ist2 whereas inheritance of ER into daughter cells is not affected. Partial overlap of Ist2 with membrane proteins of the cortical ER Since Ist2 is involved in the recruitment of ER to the PM, we investigated its localization within the cortical ER and compared it to the localization of other ER proteins. Variations in the distance between cortical ER and PM have been reported [4]. Cortical ER domains with Ist2 may represent specific sites in close contact to the PM. Genomic integration of GFP-Ist2 or mCherry-Ist2 in ist2D rescued the delayed transition from stationary into log phase, indicating that GFP- and mCherry-Ist2 proteins are functional (Fig. S4A). The surface views of mCherry-Ist2 showed localization in interconnected patch-like structures (Fig. 4). This network surrounded distinct areas free of Ist2 and other ER proteins. Such localization is typical for proteins in the cortical ER [30]. The mCherry-Ist2 signals (magenta in merged images in Fig. 4) partially overlapped with the artificial luminal ER marker GFP- HDEL, with the tail-anchored protein GFP-Ubc6, with Sec63- GFP, and with Yop1-GFP (green in merged images). The PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e39703