Finally, we tested whether the expression of human ANO1 under control of the IST2 untranslated regions rescues the delayed growth observed for ist2D after dilution from pre-cultures with OD600.2.0. Although we failed to detect the expression of ANO1 in yeast by western blotting using ANO1-specific antibody (Fig. S6E), we could observe a partial rescue (Fig. 7E). An activity at very low expression levels would be consistent with the function of Ist2 and ANO1 as ion channels. A few copies of these proteins would be able to affect the ion composition of a small volume such as the membrane-enclosed cytosol between the PM and the cortical ER. When ist2D cells were grown to an OD600 of ,3.0 (Fig. 7E, late log and transition), the integration of ANO1 rescued the delayed adaptation to log phase growth. These cells reached 111620% and 110621% of the WT growth, respectively (Fig. 7E, p,0.05). Under these conditions ist2D reached 5062% and 4467%, respectively. After dilution from stationary pre-cultures with an OD600 of .3.0 this rescue was not observed. The addition of the CSS from Ist2 to ANO1 did not improve its ability to rescue (Fig. 7E). Expression of a N-terminally mCherry-tagged, truncated version of Ist2, lacking the N-terminal domain and TMDs 1–6 (amino acids 1–477 are deleted; mCherry-Ist2D1–477 in Fig. 7E), in the cortical ER (Fig. S6F) partially rescued the ist2D phenotype (Fig. 7E). This suggests that the Ist2 function comprises both recruitment of ER to the PM and a function as ion channel. Taken together, our findings suggest a role of Ist2 in the recruitment of ER to the PM and in the functional coupling of ion transport processes between these membranes. Discussion We found that the membrane protein Ist2 recruits ER to the + PM. Ist2 activity in the cortical ER modulates glucose-induced H - pumping across the PM. Since Ist2 is required for rapid growth after dilution from pre-cultures with an OD600.2.0, Ist2-mediated coupling of ER and PM may be involved in the adaptation of cells to changing growth conditions. Ist2 is an integral membrane protein, the vast majority of which localizes to the cortical ER [5,6]. Localization of Ist2 to specific cortical ER domains is supported by a strong but not complete overlap with other ER proteins at the cell periphery. Yop1 is concentrated at edges of cortical ER in regions with high membrane curvature [30]. Like Ist2 it showed only partial overlap with other ER proteins. However, Ist2 and Yop1 did not localize to identical domains, suggesting an accumulation in distinct subdomains of the cortical ER. As an integral component of the ER membrane, Ist2 tightly connects peripheral ER structures with the PM. This may be achieved by direct binding of Ist2 to phosphoinositide lipids at the cytosolic face of the PM or by the recruitment of other factors that are involved in the association of ER and PM. The abundance of Ist2 determines the amount of cortical ER that is associated with the PM and sets the distance between cortical ER and the PM. Ist2 may function locally, since its mRNA is transported as ribonuclear particle to the bud tip and expression in the bud depends on mRNA transport [14,28]. This may explain why the function of Ist2 depends critically on the abundance, when Ist2 is expressed outside the native genomic locus. Since cortical ER structures less closely associated with the PM remain in ist2D cells, factors other than Ist2 must contribute to the formation of cortical ER. Strong overexpression of Ist2 resulted in the proliferation of cortical ER covering the PM almost completely. This overexpression of IST2 mRNA may saturate mRNA transport and lead to accumulation of Ist2 at the perinuclear ER and to alignment of the nucleus with the PM. Deletion of Ist2 did not result in defects of ER inheritance into Ist2 Mediates ER-Plasma Membrane Contacts daughter cells. However, binding of newly synthesized Ist2 to PM lipids may transform inherited ER in the daughter cell into cortical ER that is closely associated with the PM. Such conversion of ribosome-covered ER into cortical ER, with ribosomes restricted to the cytosolic face, is supported by recent electron tomography experiments [4]. Deletion of IST2 reduced the amount of cortical ER and increased the distance between ER and PM, so that ribosomes gain access to the space between ER and PM. In WT the space between cortical ER and PM lacks ribosomes. A similar ribosome- free space has been described for thin cortical ER in HeLa cells expressing STIM1 protein [48]. Therefore, this space may represent a specific microenvironment with a yet unknown protein and ion composition that is functionally separated from the surrounding cytosol [4]. Ion depletion effects in a small membrane-enclosed space have been discussed and investigated for the synaptic cleft found in the mammalian nervous system. Indeed, extensive activation of neurotransmitter-receptors in the postsynaptic membrane can deplete extracellular calcium ions in the cleft [49]. This is of conceptual interest because (depending on the synapse studied) the dimensions of the cleft between a pre- and a postsynaptic membrane can be comparable to the dimensions of the Ist2-dependent space between cortical ER and PM described here. Consistent with the idea of a functional coupling between ER and PM in tightly apposed domains we found that loss of IST2 led to increased acidification of the cytosol under glucose-starvation and to slower alkalinization in response to glucose stimulation. A function of Ist2 as an ion channel could influence cytosolic pH (pHc), which may control pH-sensitive kinases modulating Pma1 activity [41]. Ist2-dependent association of ER with the PM may contribute either directly or indirectly to glucose-induced activa- tion of Pma1 in glucose-starved cells. A regulatory role of Ist2 in the control of Pma1 activity is supported by a negative genetic interaction between ist2D and yck1D double mutants under conditions that require low Pma1 activity. Yck1 is one of two PM-bound casein kinases I in yeast that phosphorylate and down- regulate Pma1 under glucose starvation [41]. Whereas the measurement of pHc revealed a stimulatory function of Ist2 on Pma1 activity, the genetic analysis suggested an inhibitory function. These contradictory findings may be explained by differences in Pma1 activation by glucose [33]. In contrast to the measurements of pHc, where glucose-starved cells were exposed to glucose, the growth of double mutants was analyzed in constant presence of glucose. Analysis of growth at pH 3 clearly shows that Pma1 function is generally intact in ist2D cells. How can Ist2 influence the rate of H+ pumping under different environmental challenges? The association of Ist2 containing ER with Pma1 containing domains of the PM may create a specific cytosolic microcompartment with a small volume. In this microcompartment transport processes across the ER membrane and the buffering capacity of both membranes may become relevant to local pH and hence secondary active transport processes. In mammalian cells the primary ion gradients across the PM are established by the Na,K-ATPase and a direct coupling between Na,K-ATPase activity and amino acid transport has been described [50]. Besides Ist2-dependent association of ER with Pma1 containing domains of the PM, we observed that a certain area of MCP remained free of underlying cortical ER. These domains may function in processes like endo- and exocytosis, which depend on a free access to the PM from the cytosol and vice versa. Consistently, recent data showed that the cortical ER is dynamic but segregates from endocytosis initiation sites and the MCC [20,24]. Both, the expression of human ANO1 in ist2D cells PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e39703