the Pma1-GFP network (Fig. 5E, F). Most of the Sur7-mCherry domains were adjacent to or separated from the network-like GFP- Ist2 structures (Fig. 5C, D, merged images and intensity profiles). Since GFP-Ist2 signal covered 52% of the cell surface (Fig. S5E), one would expect that half of the Sur7 domains overlap with Ist2 if the distribution is random. However, only 18% of single Sur7- mCherry domains were colocalized (Fig. 6H), suggesting a non- random distribution. This is consistent with a random distribution of MCC in areas devoid of cortical ER [20]. Next, we analyzed if the proportion of the ER that colocalized with Pma1 and Sur7 changes in ist2D and pTEF1-IST2 cells. As expected in cells overexpressing Ist2, we observed a proliferation of Sec63-GFP containing cortical ER (Fig. 6C and F). In these cells the cortical ER covered 79% of the cell surface, compared to 37 and 59% in ist2D and WT cells, respectively (Fig. S5E). Changes in Ist2 expression had only minor effects on the number of MCC domains (Fig. 6G). In WT and ist2D most of the Sur7-mCherry domains were adjacent to or separated from the network-like Sec63-GFP structures, as previously seen for Ist2 in WT (Fig. 6D– F, H). In ist2D the colocalization of Sur7-mCherry and Sec63-GFP decreased, whereas it increased in pTEF1-IST2 (Fig. 6H). More- over, overexpression of Ist2 led to reduced segregation of Sur7- mCherry and Sec63-GFP. In ist2D and pTEF1-IST2 strains the number of colocalized Sur7-mCherry domains was smaller than expected from the surface area covered with Sec63-GFP (Fig. 6H and S5E). This suggests that the distribution of Sur7 domains and cortical ER is not random. Most of the Sur7 domains are separated from or adjacent to the cortical ER and this specific distribution does not require Ist2. Ist2 Mediates ER-Plasma Membrane Contacts Ist2 is required for rapid glucose-induced Pma1 activation If Ist2 functions at ER-PM contact sites, it is possible that such function influences metabolic processes at the PM. Pma1- mediated pumping of H+ across the PM is such a candidate process. It controls the cytosolic pH and is involved in the acidification of the growth medium. The activity of Pma1 consumes at least 20% of the cellular ATP and is subject to strict regulation by extracellular glucose [31,32,33]. In order to analyze the effect of Ist2 on Pma1 activity, we measured the alkalinization of the cytosol in response to Pma1 activation by glucose (Fig. 7A). Dynamic changes of the cytosolic pH in living cells expressing the pH-sensitive GFP derivate ratiometric pHluorin were measured in a plate reader [34]. In response to glucose starvation the cytosolic pH (pHc) decreased to 6.77 and 6.71 in WT and pTEF1-IST2 (Fig. 7B). This pHc decrease was more pronounced in ist2D (pH 6.63). The pHc of the PMA1 hypomorph pma1-007 was 6.08, consistent with a reduced amount of Pma1 in these cells [35]. Except for pma1-007 addition of glucose (Fig. 7B, indicated with an arrow) resulted in rapid decrease of pHc, most likely, a consequence of H+ production by glycolysis (see cartoon Fig. 7A). About one min after glucose addition the cytosol was alkalinized to a constant pHc of 7.1–7.2 within five min (Fig. 7B). Alkalinization depends on glucose- mediated activation of Pma1. Addition of galactose to glucose- starved cells did not stimulate Pma1 activity (Fig. S6A). Importantly, the amount of Ist2 expression influenced this rate of pHc change. Deletion of IST2 led to significantly slower Figure 6. Ist2 dependent proliferation of the cortical ER does not alter PM compartmentation. (A–F) Surface views taken by epifluorescence microscopy showing ist2D, WT and pTEF1-IST2 cells coexpressing Pma1-mCherry (A–C) or Sur7-mCherry (D–F) and Sec63-GFP. The merged images show Pma1-mCherry and Sur7-mCherry in magenta and Sec63-GFP in green. The fourth panels show intensity profiles of the indicated cross sections in the merged images. (G) Mean number of Sur7-mCherry maxima (black) and Pma1-mCherry minima (white) in ist2D, WT and pTEF1-IST2 cells. Error bars depict s.d.. (H) Quantification of single Sur7-mCherry domains separated from (grey), adjacent to (black), and colocalized with cortical ER (white). Corresponding GFP-labelled ER proteins and strains are indicated. doi:10.1371/journal.pone.0039703.g006 PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e39703