0.6) and large (L; .0.6). (B–D) Representative z-stack CLS images (pinhole settings of 2 airy units) of ist2D, WT and pTEF1-IST2 cells with small (B), medium (C) and large (D) daughters expressing both Yop1-mCherry (left panels; magenta in merge panels) and Sec63-GFP (middle panels; green in merge panels). (E) Immuno- blot analysis of 5.0 OD600 membranes from ist2D, WT and pTEF1-IST2 cells. Yop1-GFP (upper panel) and Sec61 (lower panel) were detected by GFP and Sec61 specific antibodies. Quantifications are shown as mean 6 s.d. (n = 4). (TIF) Figure S4 Membrane proteins localize in distinct domains of the cortical ER. (A) GFP-Ist2 and mCherry-Ist2 rescue the growth phenotype of ist2D. WT (black), ist2D (grey) and ist2D cells with either genomically integrated GFP-IST2 (green) or mCherry-IST2 (red) were diluted to 0.05 OD600 from stationary pre- cultures in complete HC media and incubated at 25uC. The relative growth after 10 hours is shown with WT set to 100% 6 s.d. (n = 4). (B) Table showing Pearson’s coefficients of colocaliza- tion of indicated proteins tagged with GFP or mCherry. Mean values 6 s.d. were quantified from indicated numbers of cells. Pairs of GFP or mCherry-tagged proteins were expressed in WT cells. (C–H) Surface views of WT cells expressing both dsRed- HDEL (C, E and G; magenta in merge and profiles) or Yop1- mCherry (D, F and H; magenta in merge and profiles) and GFP- HDEL (C and D; green in merge and profiles), Sec63-GFP (E and F; green in merge and profiles) or GFP-GFP-Ubc6 (G and H; green in merge and profiles) analyzed by epifluorescence microscopy. Intensity profiles of the indicated regions (white in merge panels) are shown in right panels. The scale bar corresponds to 2 mm. (TIF) Figure S5 Sur7 is partially separated from cortical ER. (A–D) Surface views of single cells imaged by CLS microscopy. (A) WT cells expressing Sur7-mCherry and GFP-Ist2. (B) WT cells expressing Sur7-mCherry and Sec63-GFP. (C) ist2D cells express- ing Sur7-mCherry and Sec63-GFP. (D) pTEF1-IST2 cells expressing Sur7-mCherry and Sec63-GFP. (E) Amount of cell surface covered with Sur7-mCherry (in magenta) and GFP-Ist2 or Sec63-GFP (in green) in the indicated strains calculated from the cells in A–D. (TIF) Figure S6 Ist2 function is connected to pH homeostasis. (A) Pma1 activation depends on glucose. Kinetic pH measurement References 1. Prinz WA, Grzyb L, Veenhuis M, Kahana JA, Silver PA, et al. (2000) Mutants affecting the structure of the cortical endoplasmic reticulum in Saccharomyces cerevisiae. J Cell Biol 150: 461–474. 2. Pichler H, Gaigg B, Hrastnik C, Achleitner G, Kohlwein SD, et al. (2001) A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids. Eur J Biochem 268: 2351–2361. 3. Schuck S, Prinz WA, Thorn KS, Voss C, Walter P (2009) Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response. J Cell Biol 187: 525–536. 4. West M, Zurek N, Hoenger A, Voeltz GK (2011) A 3D analysis of yeast ER structure reveals how ER domains are organized by membrane curvature. J Cell Biol 193: 333–346. 5. Maass K, Fischer MA, Seiler M, Temmerman K, Nickel W, et al. (2009) A signal comprising a basic cluster and an amphipathic alpha-helix interacts with lipids and is required for the transport of Ist2 to the yeast cortical ER. J Cell Sci 122: 625–635. 6. Fischer MA, Temmerman K, Ercan E, Nickel W, Seedorf M (2009) Binding of plasma membrane lipids recruits the yeast integral membrane protein Ist2 to the cortical ER. Traffic 10: 1084–1097. 7. Hartzell HC, Yu K, Xiao Q, Chien LT, Qu Z (2009) Anoctamin/TMEM16 family members are Ca2+-activated Cl- channels. J Physiol 587: 2127–2139. Ist2 Mediates ER-Plasma Membrane Contacts of glucose-starved cells after addition of 2% galactose. Cells were starved for glucose for 1 hour in synthetic media. At the indicated time 2% galactose was added (marked with arrow). (B) Immunoblot analysis of 5.0 OD600 membranes from ist2D, WT, pTEF1-IST2 and pma1-007 cells. Pma1 (upper panel) and Sec61 (lower panel) were detected by Pma1 and Sec61 specific antibodies. Quantifications are shown as mean 6 s.d. (n = 9). (C) Genetic interaction between ist2D, btn1D and btn2D mutants. Growth of five-fold serial dilutions of the indicated strains on plates with YPD and YDP+1 M NaCl. (D) Genetic interaction between ist2D and mutants lacking genes involved in Ca2+ homeostasis and signaling. Growth of serial dilutions of the indicated strains on plates with YPD and YDP+300 mM NaCl with an adjusted pH of 3.0 or 7.5. (E) Transformation of ist2D cells with plasmids encoding ANO1 did not result in the detection of ANO1 protein. Immunoblot analysis of 5 OD600 membranes from ist2D cells or ist2D cells transformed with an ANO1-encoding plasmid and lysate from HEK293 cells or HEK293 cells transfected with a GFP-ANO1-encoding plasmid. ANO1 protein and Sec61 were detected with ANO1-, GFP- and Sec61-specific antibodies. (F) Z- stack CLS images of ist2D transformed with a N-terminally mCherry-tagged truncated version of Ist2, lacking the N-terminal domain and TMDs 1–6 (amino acids 1–476 are deleted) in surface (left panel) and equatorial (middle panel) and phase contrast (PC; right panel) views are shown. (TIF) Plasmids used in this study. Table S1 (DOC) Acknowledgments We are grateful to B. Morgan and T. Dick for help with the pH measurements and T. Levine for plasmids. We thank K. Meese for experimental support and D. Riedel, D. Wenzel and G. Heim from the EM facility at the MPIbpc for their technical advice, P. Eggensperger and B. Morgan for comments on the manuscript and B. Dobberstein for his generous support. Author Contributions Conceived and designed the experiments: WW AK B. Schrul B. Schwappach MS. Performed the experiments: WW AK B. Schrul. Analyzed the data: WW AK B. Schrul HL. Wrote the paper: WW AK B. Schrul HL B. Schwappach MS. 8. Schroeder BC, Cheng T, Jan YN, Jan LY (2008) Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell 134: 1019–1029. 9. Yang YD, Cho H, Koo JY, Tak MH, Cho Y, et al. (2008) TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature 455: 1210–1215. 10. Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, et al. (2008) TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science 322: 590–594. 11. Suzuki J, Umeda M, Sims PJ, Nagata S (2010) Calcium-dependent phospholipid scrambling by TMEM16F. Nature 468: 834–838. 12. Ercan E, Momburg F, Engel U, Temmerman K, Nickel W, et al. (2009) A conserved, lipid-mediated sorting mechanism of yeast Ist2 and mammalian STIM proteins to the peripheral ER. Traffic 10: 1802–1818. 13. Lavieu G, Orci L, Shi L, Geiling M, Ravazzola M, et al. (2010) Induction of cortical endoplasmic reticulum by dimerization of a coatomer-binding peptide anchored to endoplasmic reticulum membranes. Proc Natl Acad Sci U S A 107: 6876–6881. 14. Juschke C, Ferring D, Jansen RP, Seedorf M (2004) A novel transport pathway for a yeast plasma membrane protein encoded by a localized mRNA. Curr Biol 14: 406–411. 15. Juschke C, Wachter A, Schwappach B, Seedorf M (2005) SEC18/NSF- independent, protein-sorting pathway from the yeast cortical ER to the plasma membrane. J Cell Biol 169: 613–622. PLoS ONE | www.plosone.org 12 July 2012 | Volume 7 | Issue 7 | e39703