and the expression of truncated Ist2, lacking the first six TMDs, led to a partial rescue of the ist2D phenotype. This suggests that Ist2 has two different functions: One as channel and another one in the recruitment of cortical ER to the PM. Since ANO1 functions as a channel a flux of anions through Ist2 in either direction across the ER membrane is possible. Further insights into the subcellular distribution of chloride [51] and putative chloride gradients across yeast intracellular membranes are required to tackle the integration of ion transport processes across the ER membrane. A flux of anions out of the cytosolic microcompartment between cortical ER and PM may reduce the electrogenic aspect of Pma1 function: Proton pumping leads to a strong hyperpolarization of the membrane. A removal of negative charges would enhance the ability of the Pma1 pump to transport protons against the force of the electric field. Secondary uptake of nutrients may depend on such a transient modulation of ion composition in the space between cortical ER and PM. More efficient nutrient uptake may assist the adaptation to growth under changing environments. Alternatively to an ion channel-based function, Ist2 may influence the lipid composition of the cortical ER or the PM. This hypothesis is raised by the recently described role of PM-localized ANO6 in asymmetric phospholipid distribution [11]. Changes of the lipid composition or protonation state at ER-PM contact sites could explain an influence of Ist2 on the localization and activity of Pma1. Decreased cytosolic pH was shown to result in release of the inositol transcription factor Opi1 from phosphatidic acid at ER- PM contact sites [52]. We found that invaginations of the PM, corresponding to the MCC, were often separated from or adjacent to the underlying cortical ER, marked by Ist2 and Sec63. This segregation between MCC and cortical ER has been observed previously [20,24]. Interestingly, this distribution was maintained under conditions with changing amounts of cortical ER. The MCC contains a number of amino acid transporters including its defining member, the arginine permease Can1 [22,23,25]. Amino acid/H+ sympor- ters of the MCC are separated from Pma1 in the PM [24]. This separation may help to locally couple specific ion transport processes in different domains of the PM. Impaired uptake of amino acids and other nutrients may explain the growth delay of ist2D cells during the adaptation to changing growth conditions. In summary, we found that Ist2 recruits ER to the PM. Close association of ER and PM may contribute to specific metabolic functions of the PM. Materials and Methods Yeast strains, media and plasmids All experiments were performed in BY4741/42 strain back- ground [53]. The IST2 gene was replaced by a natNT2 resistance gene in BY4742 using a PCR-based tagging method resulting in MSY399 [54]. Similarly, the genes PMA1, SUR7 and YOP1 were tagged by chromosomal integration of PCR amplified cassettes coding for either GFP or mCherry [54]. For constitutive overexpression of IST2 we integrated BsiWI digested pMS623 into the his3D1 locus of BY4742 resulting in MSY453. Isogenic WT (MSY474) and IST2 deletion mutant (MSY483) were generated by integration of BsiWI digested pRS303 [55] into BY4742 and MSY399, respectively. Deletion of IST2 (MSY399) was combined with btn1D, btn2D, pmr1D, cnb1D, yck1D, and hrk1D from the BY4741 deletion library by standard genetics [56]. As pma1-007 hypomorph we used the YGL007w strain from the BY4741 deletion library. The preparations of synthetic HC media and yeast transformation were carried out as described [15]. The Ist2 Mediates ER-Plasma Membrane Contacts pH of YPD plates was adjusted with 50 mM 2-(N-morpholi- no)ethanesulfonic acid (MES) and KOH and HCl. Plasmid constructions are described in table S1. TIRF microscopy Yeast cells expressing fluorescent protein tagged proteins were grown overnight at 25uC in HC media to mid log phase and immobilized on cover glasses with Concavanalin A (Sigma, Steinheim, Germany; lot# 080M7680V). Cover glasses were incubated with Concanavalin A for 1 min, dried for 1 min, and washed twice with water. 0.2 OD600 yeast culture was placed on coated cover glasses for 15 to 30 min, washed once with the respective media and mounted on glass slides with a thin layer of medium. For imaging an Olympus IX81 CellTirf microscope equipped with a 10061.4 numerical aperture (NA) oil objective lens (Olympus) and an ImagEM (C9100-13) camera (Hamamatsu) driven by Olympus xcellence software was used. GFP and mCherry fluorescence were excited with a 50 mW 488 nm diode laser (Olympus) and a 100 mW 561 nm diode laser (Cobolt, Solna, Sweden), respectively. Emission light was filtered using a multiband filter set for the appropriate wavelengths (AHF, T u¨bingen, Germany). Bright field images were acquired using DIC optics. Image processing was performed using ImageJ (http://rsb.info.nih.gov/ij/). Confocal laser scanning microscopy and image analysis Confocal imaging was performed with a Zeiss LSM 780 microscope using a 10061.40 Plan-Apochromat oil objective lens (Zeiss) and pinhole settings of 1 airy unit. GFP fluorescence was excited with the 488 nm line of the argon laser and mCherry fluorescence was excited with a 561 nm diode-pump solid-state laser. Image processing and analysis was performed using ImageJ. For area measurements, the images were analyzed by performing the following protocol. First, a mean filter of the 363 pixel neighborhood (‘smooth’) was applied to reduce pixel outlier intensities and shot noise. Second, the images were background subtracted by using identical subtraction parameters (i.e. minus 30% of maximal pixel intensity values) for all images. Third, the areas of the different image channels were measured by intensity thresholding against a background set to intensity value zero. Regions-of-interest (ROI) were applied, representing the individ- ual cell sizes, in order to measure areas and percentages of areas per whole cell size, respectively. For colocalization analysis, the set of plugins from the Wright Cell Imaging Facility for ImageJ (www.uhnresearch.ca/wcif) was applied. In short, the background-subtracted two-channel images were analyzed for colocalization at ratio settings of 20% and thresholds of zero for both channels. Areas fulfilling these colozalisation criteria were then measured against the correspond- ing Sur7 areas in order to determine the degree of colocalization. Only Sur7 domains with a maximal diameter of 500 nm that were clearly separated from neighboring domains were analyzed. The degree of colocalization was grouped and labeled as full colocalization at 81–100% of signal overlap, 21–80% as adjacent and 0–20% as separated. Epifluorescence microscopy Epifluorescence microscopy was performed with an Olympus xcellence IX81 microscope system using a 1006/1.45 Plan- Apochromat oil objective lens (Olympus) and a GFP/mCherry sbx ET filter set (AHF, Tu¨ bingen, Germany). As fluorescence light source the illumination system MT20 (Olympus) with a 150 W Xe arc burner was used. In order to colocalize Ist2 and ER proteins in the cortical ER, we acquired images of cell surfaces in focus. The PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e39703