Yeast Ist2 Recruits the Endoplasmic Reticulum to the Plasma Membrane and Creates a Ribosome-Free Membrane Microcompartment Wendelin Wolf1., Annett Kilic1., Bianca Schrul2,3., Holger Lorenz1, Blanche Schwappach2,3, Matthias Seedorf1* 1 Zentrum f u¨r Molekulare Biologie der Universita¨t Heidelberg (ZMBH), DKFZ-ZMBH-Allianz, Heidelberg, Germany, 2 Department of Biochemistry I, Universita¨tsmedizin Go¨ ttingen, Go¨ ttingen, Germany, 3 Max-Planck-Institut fu¨ r biophysikalische Chemie, Go¨ ttingen, Germany Abstract The endoplasmic reticulum (ER) forms contacts with the plasma membrane. These contacts are known to function in non- vesicular lipid transport and signaling. Ist2 resides in specific domains of the ER in Saccharomyces cerevisiae where it binds phosphoinositide lipids at the cytosolic face of the plasma membrane. Here, we report that Ist2 recruits domains of the yeast ER to the plasma membrane. Ist2 determines the amount of cortical ER present and the distance between the ER and the plasma membrane. Deletion of IST2 resulted in an increased distance between ER and plasma membrane and allowed access of ribosomes to the space between the two membranes. Cells that overexpress Ist2 showed an association of the nucleus with the plasma membrane. The morphology of the ER and yeast growth were sensitive to the abundance of Ist2. Moreover, Ist2-dependent effects on cytosolic pH and genetic interactions link Ist2 to the activity of the H+ pump Pma1 in the plasma membrane during cellular adaptation to the growth phase of the culture. Consistently we found a partial colocalization of Ist2-containing cortical ER and Pma1-containing domains of the plasma membrane. Hence Ist2 may be critically positioned in domains that couple functions of the ER and the plasma membrane. Citation: Wolf W, Kilic A, Schrul B, Lorenz H, Schwappach B, et al. (2012) Yeast Ist2 Recruits the Endoplasmic Reticulum to the Plasma Membrane and Creates a Ribosome-Free Membrane Microcompartment. PLoS ONE 7(7): e39703. doi:10.1371/journal.pone.0039703 Editor: Christopher Beh, Simon Fraser University, Canada Received April 19, 2012; Accepted May 24, 2012; Published July 9, 2012 Copyright: 2012 Wolf et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Deutsche Forschungsgemeinschaft (SE 811/5-2). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: m.seedorf@zmbh.uni-heidelberg.de . These authors contributed equally to this work. Introduction In Saccharomyces cerevisiae large areas of the plasma membrane (PM) are covered with membranes of the endoplasmic reticulum (ER) [1,2,3,4]. In a recent electron tomography study these structures have been described as PM-associated ER [4]. Here, we use the term cortical ER to describe these membranes. The cortical ER consists of interconnected tubules and cisternae, which were found at an average distance of 33 nm from the PM [2,4]. How the yeast cortical ER is formed remains an open question. The polytopic membrane protein Ist2 is a candidate for a factor involved in this process because it specifically localizes to the cortical ER [5,6]. Based on sequence homology Ist2 belongs to the TMEM16 or anoctamin (ANO) protein family [7]. Mammalian ANO1 and ANO2 have been identified as Ca2+-activated Cl2 channels [8,9,10]. ANO6 function has been implicated in Ca2+- dependent phospholipid scramblase activity [11]. In contrast to yeast Ist2 ANO1, ANO2, and ANO6 reside in the PM [8,9,10,11]. Yeast Ist2 interacts with specific PM lipids, which may contribute to the recruitment of ER to the PM. The interaction with phosphoinositide lipids requires the cortical sorting signal (CSS) at the extreme C terminus of Ist2 and leads to the accumulation of Ist2 in cortical ER domains of yeast and mammalian cells [5,6,12]. The induction of cortical ER in mammalian cells by Ist2 expression and localization to cortical ER [12,13] suggests that Ist2 is an integral part of this membrane. These recent insights argue against the model that Ist2 reaches the PM on a pathway bypassing the Golgi [14,15]. The PM is organized in domains that function in nutrient transport, signal transduction, and endo- and exocytosis [16,17,18,19,20]. In S. cerevisiae individual PM domains can be visualized by light microscopy. One of these domains, the membrane compartment of Can1 (MCC) contains the H+/arginine symporter Can1 and forms dot-like structures, which are 200–300 nm in diameter [21,22,23]. In electron microscopy (EM) these structures colocalize with furrow-like mem- brane invaginations, which are ,300 nm long [24]. Beside the defining member Can1, the MCC contains the uracil- and tryptophan/H+ symporters Fur4 and Tat2, and Nce102, as well as members of the Sur7 protein family [21,22,25,26]. Another domain, + the membrane compartment of Pma1 (MCP) contains the H - ATPase Pma1 and forms a large network-like structure [16]. Pma1 pumps H+ across the PM and thereby establishes an electrochemical gradient across the yeast PM, which drives the secondary-active uptake of nutrients, e.g. amino acids [27]. Whether and how the segregation of proteins into specific domains of the yeast PM contributes to their function remains unknown. Here we investigated the function of Ist2 at the interface of ER and PM. We found that Ist2 recruits ER to the PM. This creates a PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e39703