Ist2 Mediates ER-Plasma Membrane Contacts Figure 7. Ist2 contributes to the regulation of Pma1 activity. (A) Cartoon illustrating glucose-induced H+ pumping by Pma1. (B) Kinetic measurement of cytosolic pH in ist2D, WT, pTEF1-IST2, and pma1-007 strains. Cells were starved for glucose for 1 hour in synthetic media. At the indicated time, marked with an arrow, 2% glucose was added. (C) Quantification of the rate of H+ pumping shown as DpH/min. (D) Five-fold serial dilutions of the indicated strains on plates with YPD, YPD adjusted to pH of 3.0, and YPD+100 mg/ml hygromycin B. (E) Growth of ist2D and ist2D expressing ANO1, ANO1+CSS or mCherry-Ist2D1–477 during the first 10 hours after dilution from pre-cultures with an OD600 of 2.0–2.5 (late log), 2.5– 3.0 (transition), and .3.0 (stationary). Growth of the corresponding WT was set to 100%. In (C) and (E) error bars depict s.d., non-significant differences are indicated as n.s., and triple asterisks show significance (p,0.001). doi:10.1371/journal.pone.0039703.g007 alkalinization of the cytosol as compared to WT (p,0.05), whereas overexpression of IST2 resulted in a slightly but not significantly faster alkalinization (quantification in Fig. 7C). The amount of Pma1 did not change significantly in cells with different amounts of Ist2, whereas pma1-007 showed decreased Pma1 expression (Fig. S6B, and [35]). Thus, Ist2 influences H+ pumping in response to glucose. The activity of Pma1 is under strict control. Growth at acidic pH requires strong activation of Pma1 [35]. Cells with reduced Pma1 expression display a growth defect at pH 3.0, as seen for pma1-007 (Fig. 7D). Down-regulation of Pma1 activity also results in a reduced uptake of toxic cations [36,37]. Consequently pma1- 007 grows better than WT in the presence of a toxic concentration of the cation hygromycin B [38,39]. Deletion of IST2 led to impaired growth in media containing hygromycin B, but had no effect on growth at pH 3.0 (Fig. 7D) indicating that Pma1 function is intact. In order to further test whether Ist2 function is required under growth conditions where Pma1 activity is regulated, we combined ist2D with the deletion of YCK1 and tested growth on hygromycin B and at low pH. Yck1 is a PM-bound casein kinase I that down-regulates Pma1 activity in response to glucose starvation [40,41]. The double mutant showed an enhanced growth defect on hygromycin B, whereas growth on YPD and on YPD pH 3.0 was similar to WT (Fig. 7D). This synthetic growth defect was specific, as the deletion of the positive Pma1 regulator Hrk1 kinase [42] had no effect (Fig. 7D). The observed negative genetic interaction between IST2 and YCK1 is consistent with the hypothesis that presence of Ist2 influences Pma1 activity. Another study reported a negative genetic interaction between IST2 and BTN1 or BTN2 double mutants at high NaCl concentra- tion [43]. Btn1 is the yeast orthologue of human CLN3, which is involved in neuronal ceroid lipofuscinosis characterized by an accumulation of storage material in lysosomes [44]. Btn2, which is up-regulated in BTN1 mutants, functions as a SNARE and retromer-interacting protein that is involved in the recycling of cargo proteins from late endosomes to the Golgi [45]. In order to clarify this aspect of Ist2 function, we analyzed the growth of ist2D, btn1D and btn2D single and double mutants at 1 M NaCl concentration. Only the single btn2D mutant showed a slight growth defect on YPD plates with 1 M NaCl, whereas all other mutants grew at least as well as the WT (Fig. S6C). This was independent of LYS2 and MET15 genotypes in BY4741 and BY4742 strains. Our results contradict the previously reported growth defect of ist2D/ btn1D and ist2D/btn2D double mutants on high salt [43]. The putative mammalian Ist2 orthologues ANO1 and ANO6 function as a Ca2+-activated Cl2 channel or are involved in phospholipid scramblase activity, respectively [8,9,10,11]. There- fore, we tested whether changes in Ca2+ homeostasis or signaling had an influence on the growth of IST2 mutants. The Ca2+ ATPase Pmr1 pumps Ca2+ from the cytosol into ER and Golgi and its deletion results in increased cytosolic Ca2+ concentration [46]. Double deletion of PMR1 and IST2 led to a salt-dependent growth defect at acidic pH (Fig. S6D). In order to interfere with Ca2+ signaling, we deleted CNB1, the regulatory subunit of the Ca2+/calmodulin-regulated type 2B protein phosphatase calci- neurin [47]. This deletion resulted in a salt- and alkaline pH- dependent growth defect but additional deletion of IST2 did not change this phenotype (Fig. S6D). Hence by employing growth assays we were not able to uncover interplay between IST2 function and Ca2+ signaling. PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e39703