Golgi Ca2+ storage

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Ca2+ homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca2+ storage Petra D’hooge a , Catherina Coun b , Vincent Van Eyck a , Liesbeth Faes a , Ruben Ghillebert b , Lore Mariën a , Joris Winderickx b,∗ , Geert Callewaert a,∗∗ a b

The Yeast Hub Lab, KU Leuven, Campus Kulak, Etienne Sabbelaan 53, 8500 Kortrijk, Belgium Functional Biology, Department of Biology, KU Leuven, Kasteelpark Arenberg 31, 3001 Heverlee, Belgium

a r t i c l e

i n f o

Article history: Received 10 February 2015 Received in revised form 11 May 2015 Accepted 26 May 2015 Available online xxx Keywords: Calcium homeostasis Vacuole ER Golgi Saccharomyces cerevisiae

a b s t r a c t Yeast has proven to be a powerful tool to elucidate the molecular aspects of several biological processes in higher eukaryotes. As in mammalian cells, yeast intracellular Ca2+ signalling is crucial for a myriad of biological processes. Yeast cells also bear homologs of the major components of the Ca2+ signalling toolkit in mammalian cells, including channels, co-transporters and pumps. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar Ca2+ transporters, combined with manipulations to estimate intracellular Ca2+ storage, we evaluated the contribution of individual transport systems to intracellular Ca2+ homeostasis. Yeast strains lacking Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca2+ homeostasis, displayed a fragmented vacuolar phenotype and showed increased vacuolar Ca2+ uptake and Ca2+ influx across the plasma membrane. In the pmr1 strain, these effects were insensitive to calcineurin activity, independent of Cch1/Mid1 Ca2+ channels and Pmc1 but required Vcx1. By contrast, in the cod1 strain increased vacuolar Ca2+ uptake was not affected by Vcx1 deletion but was largely dependent on Pmc1 activity. Our analysis further corroborates the distinct roles of Vcx1 and Pmc1 in vacuolar Ca2+ uptake and point to the existence of not-yet identified Ca2+ influx pathways. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction As in all eukaryotic cells, yeast cells employ calcium signalling mechanisms to regulate a wide variety of cellular processes including cell-cycle progression, mating, protein processing, responses to hypotonic stress, maintenance of intracellular pH [1–8] and adequate nutritional and metabolic signalling [9–12]. Moreover, the toolkit of Ca2+ signalling proteins in yeast and mammalian cells is remarkably similar. Cytosolic free Ca2+ concentration in yeast cells is delicately regulated and maintained at low levels (50–200 nM) through the action of different Ca2+ transporters including channels, co-transporters and pumps and some of these Ca2+ transporters are evolutionarily conserved from yeast to human [13–15]. Extracellular Ca2+ can enter the cytosol through several Ca2+ influx pathways. The best characterized pathway is a high affinity, low capacity influx system composed of Cch1 and Mid1, the yeast

∗ Corresponding author. Tel.: +32 16 321516 ∗∗ Corresponding author. Tel.: +32 56 246224. E-mail addresses: [email protected] (J. Winderickx), [email protected] (G. Callewaert).

homologue of voltage-gated Ca2+ channels [16–23]. In fungal and yeast cells the Cch1/Mid1 complex, however, primarily functions as a store-operated Ca2+ channel that is activated upon depletion of secretory Ca2+ [18,24]. Several other Ca2+ influx pathways have been reported, but are not fully characterized at the molecular level yet. These include a low affinity, high capacity Ca2+ channel involved in pheromone-signalling [4,7], two transporters referred to as X and M involved in Ca2+ influx in non-stimulated yeast cells grown under standard conditions [13] and a glucose-induced Ca2+ influx or GIC pathway that provides influx when Cch1/Mid1 channels are inactivated [25]. Following Ca2+ influx, cytosolic Ca2+ is mainly transported into the vacuole, the topologically equivalent of the mammalian lysosome [26]. The vacuole contains approximately ≥95% of total cellular Ca2+ , largely in complex with inorganic polyphosphate. Vacuolar Ca2+ sequestration is accomplished by the Ca2+ ATPase Pmc1 [27,28] and the Ca2+ /H+ exchanger Vcx1 [29]. Upon hypotonic and mechanical stress [30,31], Ca2+ can be released from the vacuole through the Yvc1 channel, a homolog of mammalian TRPC-type Ca2+ channels [32]. In addition to the vacuole, the ER and Golgi apparatus also play a crucial role in maintaining proper Ca2+ homeostasis in yeast cells. Three transporters, the Ca2+ -ATPase Pmr1 [33] and presumable the ATPase Cod1 [34] and the Ca2+ /H+ exchanger Gdt1 [35,36] function together in ER/Golgi

http://dx.doi.org/10.1016/j.ceca.2015.05.004 0143-4160/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: P. D’hooge, et al., Ca2+ homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca2+ storage, Cell Calcium (2015), http://dx.doi.org/10.1016/j.ceca.2015.05.004

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Ca2+ sequestration. Pmr1 is a Secretory Pathway Ca2+ ATPase (SPCA) that transports Ca2+ and Mn2+ into the secretory pathway compartments. Transport of Ca2+ (and Mn2+ ) into the lumen of ER/Golgi is crucial for the correct folding and processing of proteins during their transport through the secretory pathway [37–39]. Re-addition of glucose to carbohydrate starved yeast cells results in a variety of effects including increased turnover of phosphatidyl inositol, activation of the Ras-adenylate cyclase pathway [40], activation of the plasma membrane H+ -ATPase [41], intracellular acidification due to glycolysis, which leads to the activation of the vacuolar H+ ATPase [9,42,43] and a transient elevation of cytosolic Ca2+ or TECC response [10,12]. This TECC response has been shown to depend on glucose uptake and phosphorylation [11]. Despite extensive knowledge about the molecular identity and individual function of most Ca2+ transporters, we are still far from a full understanding of the complexity that governs Ca2+ homeostasis. In this study, we extend previous studies by analysing Ca2+ responses in Saccharomyces cerevisiae expressing the Ca2+ dependent photoprotein aequorin. The relative importance of individual Ca2+ transport systems was evaluated using single- and multiplegene deletion strains of Ca2+ transporters in conjunction with a novel experimental approach to estimate intracellular Ca2+ storage. We show that deletion of the ER/Golgi transporters Pmr1 and Cod1 promotes vacuolar fragmentation and markedly enhances Ca2+ storage and Ca2+ influx whereas deletion of the vacuolar transporters Vcx1 and Pmc1 leads to an overall decrease in Ca2+ storage.

2. Materials and methods 2.1. Yeast strains, plasmids, and media In this study we used the yeast S. cerevisiae BY4741 (Mata his31 leu20 met150 ura30) and different deletion strains (Euroscarf, Frankfurt, Germany). Multiple deletion strains were generated by standard genetic crossing, and verified by PCR. YPD medium containing 2% peptone, 1% yeast extract and 2% glucose was used for growth and maintenance of yeast cells. Synthetic complete medium (Sc) containing 0.19% yeast nitrogen base without amino acids, 0.5% ammonium sulphate supplemented with synthetic drop-out amino-acid (Leu and Ura)/nucleotide mixture and 2% glucose (SD) was used for the selection, growth and maintenance of transformed yeast strains. Transformation of yeast cells was performed following the lithium/polyethylene glycol method [44]. To monitor cytosolic Ca2+ levels, strains were transformed with pYX212 vector encoding cytosolic aequorin (pYX212-cytAEQ) (kind gift from E. Martegani, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy) [45]. Furthermore, in order to use the yeast strains as a model to study the effects of expression of heterologous proteins on Ca2+ homeostasis at later stages, all yeast strains were now already transformed with an empty control vector (pGGE181-EV).

2.2. Determination of growth profiles To evaluate the effect of the different deletions on growth, the growth profiles of yeast were determined (Sup. Fig. 1). All yeast strains transformed with the pYX212-cytAEQ and PGGE181-EV were grown overnight. These cultures were used to inoculate new cultures at a starting OD600 of 0.05. Growth was quantified by measuring the OD600 over 70 h period (plate reader Multiskan Go, Thermo Scientific). Absorbance data were averaged across at least 18 replicates for each strain and plotted as a function of time.

2.3. Cytosolic Ca2+ measurements using aequorin Cytosolic Ca2+ levels ([Ca2+ ]in ) were measured in populations of yeast cells expressing aequorin essentially as described [46,47]. Briefly, yeast cells were transformed with the pYX212 encoding apoaequorin gene under the control of the TPI promoter and the pGGE181-EV and grown in selective medium with 2% glucose. Cells taken from stationary-phase pre-cultures were used to inoculate a new culture. When cultures reached an OD600 of ±2–3, two OD600 units of cells were plated on concanavaline A coated coverslips and incubated at 30 ◦ C for 1 h. Cells were subsequently washed with 0.1 M 2-(N-morpholino) ethanesulphonic acid (MES)/Tris, pH 6.5, which is a nutrient free buffer, and again incubated for 1 hr at 30 ◦ C with 0.1 M MES/Tris pH 6.5 supplemented with 5 ␮M wild-type coelenterazine (Promega) to charge aequorin. Excess of coelenterazine was removed by washing the cells 3 times with 0.1 M MES/Tris pH 6.5 and coverslips were mounted in a thermostated perfusion chamber (30 ◦ C). Glucose-starved yeast cells were initially perfused with 0.1 M MES/Tris pH 6.5, followed by 0.1 M MES/Tris pH 6.5 supplemented with 10 mM CaCl2 (referred to as Ca2+ pulse). Cells were then stimulated by addition of 80 mM glucose to induce a transient elevation of cytosolic Ca2+ (referred to as TECC response) [10–12]. To estimate intracellular Ca2+ storage following the Ca2+ pulse (referred to as Ca2+ pulse release) or the TECC response (referred to as TECC release), cells were exposed for 90 s to a Ca2+ -free medium containing (in mM): 200 KCl, 100 NaCl, 3 EGTA, 20 Hepes/KOH pH 6.8 and then subsequently permeabilized with 0.5% Triton X-100 in the same medium. At the end of all experiment, cells were perfused in a Ca2+ -rich hypotonic medium (10 mM CaCl2 in H2 O). Photons emitted as a result of Ca2+ binding to charged aequorin were detected by a photon-counting tube (Type H3460-04, Hamamatsu Photonics, Japan) that was positioned about 2 cm above the cells. Light impulses were discriminated, prescaled and counted with a PC-based 32-bit counter/timer board (PCI-6601, National Instruments Corporation, Austin, TX, USA). The number of impulses occurring during a 1 s time interval was monitored with custom-built software. The recorded aequorin luminescence data were calibrated offline into cytosolic Ca2+ ([Ca2+ ]in ) values using the following algorithm [Ca2+ ]in = ((L/Lmax )1/3 + [118(L/Lmax )1/3 − 1)/(7 × 106 − [7 × 106 (L/Lmax )1/3) ]) where L is the luminescence intensity at any time point and Lmax is the integrated luminescence [48]. In selected experiments, cells were preincubated for 2 or 43 hrs with 20 ␮g/ml cyclosporine A (CsA – Sigma–Aldrich) or 4 ␮g/ml FK506 (Invivogen). CsA was dissolved in ethanol, FK506 in DMSO and results were compared to cells pretreated with vehicle alone.

2.4. Yeast vacuolar staining Vacuolar membranes were labelled by GFP-tagged Vph1p, a transmembrane subunit of the vacuolar proton ATPase. GFP-Vph1p expressing cells (OD600 of ±1.2 incubated in 0.1 M MES/Tris pH 6.5 supplemented with 10 mM external Ca2+ ) were visualized with a Zeiss LSM 710 laser scanning microscope using a 100× high NA objective, and image processing was done using ZEN software. Vacuole morphology was quantified in >250 cells of each yeast strain and scored as either having one to two vacuoles or >two vacuoles.

2.5. Statistical analysis and curve fitting Ca2+ values are expressed as mean ± standard error of the mean (SEM) and n is the number of experiments performed. Comparisons between two groups were carried out using an unpaired t-test. P < 0.001 (marked by one asterisk) indicates statistical significance.

Please cite this article in press as: P. D’hooge, et al., Ca2+ homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca2+ storage, Cell Calcium (2015), http://dx.doi.org/10.1016/j.ceca.2015.05.004

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OD600 nm values are expressed as mean ± standard error of the mean (SEM) and n is the number of experiments performed. Comparisons between two groups were carried out using an unpaired t-test. P < 0.05 (marked by one asterisk) and P < 0.001(marked by two arterisks) indicates statistical significance. Linear and non-linear regression curve fitting was performed using Graphpad Prism software.

3. Results 3.1. Ca2+ responses in wild-type yeast cells The experiments shown in Fig. 1 were designed to estimate Ca2+ uptake into intracellular stores following addition of external Ca2+ or re-addition of glucose to carbohydrate starved yeast cells. Fig. 1A shows the result of an experiment in which addition of 10 mM external Ca2+ induced an immediate increase in cytosolic Ca2+ (referred to as the Ca2+ pulse) that gradually declined to a new steady-state resting level of about 0.37 ± 0.01 ␮M Ca2+ (n = 159). Thereafter, cells were briefly exposed to Ca2+ free medium and subsequently permeabilized using Triton X-100. Permeabilization of the membrane induced release of the Ca2+ sequestered during the Ca2+ pulse (referred to as the Ca2+ pulse release). As shown by the graph of the derivative in Fig. 1A, Ca2+ pulse release occurred in two phases: a small rapid increase which was followed by a slow and steadily further increase in cytosolic Ca2+ that peaked at 1.15 ± 0.07 ␮M Ca2+ (n = 159) over the next 5 min. Fig. 1B shows the result of a separate experiment in which the Ca2+ pulse was first followed by re-addition of glucose before permeabilization. Re-addition of glucose to carbohydrate starved cells led to a transient elevation of cytosolic calcium (referred to as the TECC response) with an apparent delay of about 60 s, confirming previous findings concerning TECC responses in yeast cells [10–12]. In medium containing 10 mM Ca2+ , the peak of this TECC response induced with 80 mM glucose amounted to 1.57 ± 0.07 ␮M (n = 161). Following the peak value cytosolic Ca2+ levels slowly returned to a new steady-state level of about 0.50 ± 0.02 ␮M Ca2+ (n = 161). Permeabilization after the TECC response also induced a two-phase Ca2+ release response (referred to as the TECC release): a small rapid increase in cytosolic Ca2+ which was immediately followed by a large surge in cytosolic [Ca2+ ] reaching a peak value of 2.37 ± 0.08 ␮M Ca2+ (n = 161) over the next 4 min and then slowly declining to a value of about 1.00 ± 0.08 ␮M Ca2+ (n = 3) after 20 min. In the absence of external Ca2+ , the TECC response was greatly inhibited (Fig. 1B – grey trace), indicating that the TECC response depends on extracellular Ca2+ . Comparison of the Ca2+

Fig. 1. Ca2+ responses in wild type BY4741 yeast cells. (A) Representative averaged recordings of cytosolic [Ca2+ ] as a function of time during a 2 min application of 10 mM external Ca2+ (referred to as the Ca2+ pulse). Thereafter, cells were briefly exposed to Ca2+ free intracellular medium prior to membrane permeabilization using Triton X-100 (blue arrowhead). Permeabilization produced a two phase Ca2+ pulse release signal. (B) In separate experiments, following the Ca2+ pulse, 80 mM glucose was re-added (blue bar) which produced a transient elevation of cytosolic calcium (referred to as the TECC response). Thereafter, cells were briefly exposed to Ca2+ free intracellular medium prior to membrane permeabilization using Triton X-100 (blue arrowhead). Permeabilization produced a two phase TECC release signal. Superimposed grey trace denotes TECC response in the absence of external Ca2+ . Green traces denote the first derivative of the [Ca2+ ] signal; patterned red bars perfusion with medium containing 10 mM Ca2+ ; blue bars perfusion with medium containing 80 mM glucose; blue arrowheads the time of addition of Triton X-100; and blue circles quantitatively evaluated parameters. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

pulse and TECC release clearly indicates that yeast cells store Ca2+ more efficiently when glucose is present. It is also significant that Ca2+ release consistently occurs in two phases of which only the second larger Ca2+ release is significantly enhanced when glucose is present. For TECC responses, the EC50 for glucose was reached at a dose of 18.28 mM (Fig. 2A). Increasing the external Ca2+ concentration from 10 to 50, 100 and 200 mM increased both the peak value of the Ca2+ pulse and that of the TECC response (Fig. 2B). In all subsequent experiments TECC responses were evoked using 80 mM glucose in a medium containing 10 mM external Ca2+ . To identify intracellular Ca2+ stores and Ca2+ transporter systems underlying these Ca2+ responses, we next analyzed strains

Fig. 2. Ca2+ and glucose dependence of Ca2+ responses in wild type BY4741 yeast cells. (A) In the presence of 10 mM external Ca2+ , the EC50 for glucose of the TECC response was 18.28 mM. Continuous line represents the sigmoidal fit to the experimental data (with R2 = 0.9034). Each data point is the mean ± SEM of at least 5 measurements. (B) The EC50 for external Ca2+ of the Ca2+ pulse and TECC response induced by 80 mM glucose was 137.3 and 130.5 mM, respectively. Continuous lines represent the sigmoidal fit to the experimental data (for peak Ca2+ pulse R2 = 0.9802 and ˇ = 1.346; for peak TECC response R2 = 0.906 and ˇ = 1.539). Each data point is the mean ± SEM of at least 5 measurements.

Please cite this article in press as: P. D’hooge, et al., Ca2+ homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca2+ storage, Cell Calcium (2015), http://dx.doi.org/10.1016/j.ceca.2015.05.004

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Fig. 3. Ca2+ responses in deletion strains. Representative averaged recordings of cytosolic [Ca2+ ] as a function of time in different deletion strains (black traces – deletion strain indicated at top of each column) and wt yeast (superimposed grey traces) in response to addition of 10 mM external Ca2+ (Ca2+ pulse – upper panels) and subsequent permeabilization using Triton X-100 (blue arrowhead); or a Ca2+ pulse followed by re-addition of 80 mM glucose (blue bar – TECC response – lower panels); and subsequent permeabilization using Triton X-100 (blue arrowhead). Patterned red bars indicate perfusion with medium containing 10 mM Ca2+ ; blue bars perfusion with medium containing 80 mM glucose; and blue arrowheads the time of addition of Triton X-100. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

with single or multiple deletions of various Ca2+ transport proteins. Several parameters were quantitatively evaluated from recorded data including peak [Ca2+ ] attained during the Ca2+ pulse and TECC response, [Ca2+ ] reached at the end of the TECC response, and [Ca2+ ] obtained 4–5 min after permeabilization (Fig. 1 – crossed circles).

Ca2+

3.2. responses in deletion strains of vacuolar transporters.

Ca2+

In a first step, we examined Ca2+ signals in deletion strains lacking different vacuolar Ca2+ transporters including the Ca2+ /H+ exchanger Vcx1, the Ca2+ -ATPase Pmc1 and the Ca2+ release channel Yvc1. We also constructed the double deletion strain vcx1/pmc1, lacking both vacuolar Ca2+ uptake pathways. Ca2+ responses in glucose-starved yvc1 cells were similar to those observed in wild type (wt) cells. By contrast, deletion of Vcx1, Pmc1 or Vcx1/Pmc1 greatly affected the characteristics of Ca2+ responses. Averaged recordings are shown in Fig. 3 while quantified parameters are summarized in Figs. 4–6. Firstly, in all three deletion strains the amount of Ca2+ released following a Ca2+ pulse was significantly decreased whilst amplitude and kinetics of the Ca2+ pulse were not significantly affected. The decrease in Ca2+ pulse release was more pronounced in pmc1/vcx1 and pmc1 compared to vcx1. Furthermore, TECC responses (both peak and Ca2+ levels reached at the end of the TECC response) were significantly enhanced and the amount of Ca2+ released following the TECC response significantly decreased in the vcx1 and vcx1/pmc1, but not in the pmc1 deletion strain. Finally, in the vcx1/pmc1 strain, a synergistic effect was observed and cytosolic Ca2+ levels clearly failed to fully relax in a Ca2+ free medium. These findings clearly show that deletion of Vcx1 results in decreased Ca2+ storage (both Ca2+ pulse and TECC release decreased) and hence that vacuolar Ca2+ sequestration is a major mechanism for clearing cytosolic Ca2+ . Furthermore, vacuolar Ca2+ uptake via Pmc1 is mainly operative upon addition of

Ca2+ to the medium while Vcx1 is apparently the main mechanism for Ca2+ uptake during the TECC response. 3.3. Ca2+ responses in deletion strains of Ca2+ transporters of the ER/Golgi compartment Three transporters, the Ca2+ -ATPase Pmr1 [33], the ATPase Cod1 [34] and the Ca2+ /H+ exchanger Gdt1 [35,36] function together in ER/Golgi Ca2+ sequestration. In the next experiments, we examined the effects of loss of individual transporters or combined deletion of Pmr1/Cod1 and Cod1/Gdt1. Retarded growth was observed in pmr1 and pmr1/cod1 (Sup. Fig. 1). Averaged recordings are shown in 3 while quantified parameters are summarized in Figs. 4–6. Compared to wt cells, pmr1 and cod1 cells displayed a significant larger peak TECC response concomitant with a significant increase in TECC release. Most remarkably, however, discharge of Ca2+ stores following the Ca2+ pulse caused significant larger Ca2+ pulse release signals in cells lacking either Pmr1 and/or Cod1 whilst the amplitude and kinetics of the Ca2+ pulse were not affected. Ca2+ responses in cod1/gdt1 were similar to those seen in cod1 cells and single deletion of Gdt1 had no effect except for a significant albeit small decrease in Ca2+ pulse release. These findings suggest that under the present experimental conditions Gdt1 contributes little to Ca2+ sequestration while deletion of Pmr1 and/or Cod1 dramatically increase Ca2+ storage and influx during the Ca2+ pulse. Thus, in contrast to deletion of vacuolar active Ca2+ transport systems (Vcx1 and/or Pmc1), deletion of Pmr1 and/or Cod1 results in increased Ca2+ pulse release. 3.4. Ca2+ responses in deletion strains of plasma membrane Ca2+ transporters The involvement of Cch1 or Mid1 was investigated in cch1, mid1 and cch1/mid1 strains. Unexpectedly, deletion of both these genes had only small effects on Ca2+ responses. First, Ca2+

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Fig. 4. Graph showing mean (with vertical SEM bar) measured Ca2+ pulse release in wt and deletion strains. Open black square symbol denotes wt yeast. Filled red, green and black circles denote deletion strains lacking either vacuolar, ER/Golgi or plasma membrane Ca2+ transporters, respectively. Mixed colours denote deletion strains lacking ER/Golgi and vacuolar Ca2+ transporters (green border – red centre and SEM bar) or ER/Golgi and plasma membrane Ca2+ transporters (green border – black centre and SEM bar). Number of experiments is indicated below each symbol. P < 0.001 (data with an asterisk) indicates statistically significantly different values from wt cells, determined using unpaired t-tests. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

pulse release was decreased somewhat in all three deletion strains. Second, peak TECC response and TECC release were decreased in the double mutant but not in the single deletion strains. Although the latter finding may indicate that Cch1 and its subunit Mid1 are responsible for mediating Ca2+ influx across the plasma membrane, it is overall clear that under the present experimental conditions other not yet identified Ca2+ influx pathways contribute to Ca2+ homeostasis. Thus, in comparison to the deletion of vacuolar or ER/Golgi transporters, the deletion of Cch1 and/or Mid1 had minimal effects on Ca2+ homeostasis. 3.5. Ca2+ responses in deletions strains that lack both ER/Golgi and vacuolar or plasma membrane transporters. Based on Ca2+ responses mutants generally fall in two classes: those that display increased TECC and Ca2+ pulse release and

those characterized by overall decrease in Ca2+ storage (Fig. 7). The first class, referred to as ER/Golgi phenotype, includes in addition to pmr1 and/or cod1 also cod1/gdt1, vcx1/cod1, pmc1/cod1 and pmr1/cch1/mid1. TECC release was not affected in pmc1/pmr1 cells but this strain also displayed significant increased Ca2+ pulse release. The second class, referred to as vacuole phenotype, is exemplified by the vcx1 strain and phenocopied in vcx1/pmc1 and vcx1/pmr1. TECC release was not affected in pmc1 cells but this strain also displayed significant decreased Ca2+ pulse release. Retarded growth was observed in pmc1/cod1, pmc1/pmr1 and pmr1/cch1/mid1 cells (Sup. Fig. 1). As shown in Fig. 8A, the large Ca2+ pulse release signal in pmr1 cells was not significantly affected in mutants that also lacked Pmc1 or Cch1/Mid1. In contrast, Ca2+ pulse release signal was decreased in the vcx1/pmr1 strain relative to the wild-type. Previous

Fig. 5. Graph showing mean (with vertical SEM bar) measured peak TECC response in deletion strains. Open black square symbol denotes wt yeast. Filled red, green and black circles denote deletion strains lacking either vacuolar, ER/Golgi or plasma membrane Ca2+ transporters, respectively. Mixed colours denote deletion strains lacking ER/Golgi and vacuolar Ca2+ transporters (green border – red centre and SEM bar) or ER/Golgi and plasma membrane Ca2+ transporters (green border – black centre and SEM bar). Number of experiments is indicated below each symbol. P < 0.001 (data with an asterisk) indicates statistically significantly different values from wt cells, determined using unpaired t-tests. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Graph showing mean (with vertical SEM bar) measured TECC release in deletion strains. Open black square symbol denotes wt yeast. Filled red, green and black circles denote deletion strains lacking either vacuolar, ER/Golgi or plasma membrane Ca2+ transporters, respectively. Mixed colours denote deletion strains lacking ER/Golgi and vacuolar Ca2+ transporters (green border – red centre and SEM bar) or ER/Golgi and plasma membrane Ca2+ transporters (green border – black centre and SEM bar). Number of experiments is indicated below each symbol. P < 0.001 (data with an asterisk) indicates statistically significantly different values from wt cells, determined using unpaired t-tests. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Ca2+ storage in wt and deletion strains. Graph showing relationship between TECC release and Ca2+ pulse release in wt and deletion strains. Open black square symbol denotes wt yeast. Filled red, green and black circles denote deletion strains lacking either vacuolar, ER/Golgi or plasma membrane Ca2+ transporters, respectively. Mixed colours denote deletion strains lacking ER/Golgi and vacuolar Ca2+ transporters (green border – red centre and SEM bars) or ER/Golgi and plasma membrane Ca2+ transporters (green border – black centre and SEM bars). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

studies [49] have implicated the calcineurin-dependent pathway in the over-accumulation of Ca2+ into the vacuole in pmr1 cells. However, we found that short- (2 h) or long-term (43 h) preincubation with CsA (Fig. 8B) or FK506 (data not shown), two specific inhibitors of calcineurin, further increased Ca2+ pulse release in both wild type and pmr1 cells. In contrast to pmr1 cells, the large Ca2+ pulse release signal in cod1 cells was not significantly affected in mutants that also lacked Vcx1 but significantly decreased in pmc1/cod1 cells (Fig. 8C). 3.6. Correlation vacuolar morphology and Ca2+ responses. It is well known that yeast vacuoles may undergo fusion and fission during the cell cycle and in response to changes in environmental conditions. Given that the vacuole is the main storage for Ca2+ ions in yeast cells, we also investigated whether changes in Ca2+ pulse release is paralleled by changes in vacuolar structure. Using GFP-tagged Vph1p one to two large vacuoles

were typically observed in wild-type yeast. As shown in Fig. 9, many deletion strains with ER/Golgi phenotype (increased Ca2+ pulse release) including pmr1 and/or cod1, cod1/gdt1, vcx1/cod1, pmc1/cod1 and pmr1/cch1/mid1 also displayed multiple small vacuoles.

4. Discussion In this work, we recorded Ca2+ signals in wild-type yeast and Ca2+ transporter deletion strains when external Ca2+ was added (Ca2+ pulse) or glucose re-added (TECC response) using the bioluminescent protein aequorin. To exactly estimate how much Ca2+ was stored under various conditions, cells were permeabilized with Triton X-100 in a Ca2+ -free medium supplemented with EGTA. Release of stored Ca2+ following the Ca2+ pulse or the TECC response was clearly biphasic We assume that the fast Ca2+ transient reflects release of free Ca2+ stored in organelles while the second slower component comprises slow release of bound Ca2+ .

Please cite this article in press as: P. D’hooge, et al., Ca2+ homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca2+ storage, Cell Calcium (2015), http://dx.doi.org/10.1016/j.ceca.2015.05.004

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Fig. 8. Ca2+ pulse release in Pmr1 and Cod1 deletion strains. (A) Representative averaged recordings of cytosolic [Ca2+ ] as a function of time in different Pmr1 deletion strains and wt yeast in response to addition of 10 mM external Ca2+ and subsequent permeabilization using Triton X-100. (B) Representative averaged recordings of cytosolic [Ca2+ ] as a function of time in pmr1 (red traces) and wt (black traces) yeast under control conditions (light coloured traces) and following 43 h preincubation with CsA (dark coloured traces). (C) Representative averaged recordings of cytosolic [Ca2+ ] as a function of time in different Cod1 deletion strains and wt yeast in response to addition of 10 mM external Ca2+ and subsequent permeabilization using Triton X-100. Patterned red bars indicate perfusion with medium containing 10 mM Ca2+ and blue arrowheads the time of addition of Triton X-100. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Our initial observations clearly show that in wild-type yeast more Ca2+ is stored during the TECC response than during a Ca2+ pulse. Glucose starvation leads to a significant decrease of cytosolic pH, which is rapidly restored on re-addition of glucose presumably as a result of vacuolar H+ -ATPase (V-ATPase) activation through reassembly [50,51] and activation of the plasma membrane H+ -ATPases (Pma1 and Pma2) [52]. The resulting vacuolar transmembrane pH gradient stimulates Ca2+ /H+ exchange through

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Vcx1 and Ca2+ uptake in the vacuole [53]. This scheme is consistent with our finding that in vcx1 deletion strains (vcx1, vcx1/pmc1 and vcx1/pmr1) Ca2+ storage during the TECC response is greatly attenuated. The yeast vacuole membrane also contains Pmc1, a high-affinity Ca2+ -ATPase. Single deletion of Pmc1 had no significant effect on Ca2+ storage during the TECC response but greatly attenuated Ca2+ storage during the Ca2+ pulse. These findings suggest that Pmc1 provides and maintains background vacuolar Ca2+ levels, while pH-dependent Vcx1 activity may significantly boost vacuolar Ca2+ storage. As a consequence of a general loss of vacuole Ca2+ sequestration, Ca2+ homeostasis (Ca2+ pulse release, peak TECC response and TECC release) was found most disrupted in the double mutant vcx1/pmc1. These data thus provide additional evidence that the vacuole is the major Ca2+ storage. The results further show that single deletion of Yvc1 had no effect on uptake or storage of vacuolar Ca2+ . It is noteworthy that in good agreement with the work of Groppi et al. [25] TECC responses in the absence of external Ca2+ were not completely eliminated despite the fact that cells were kept in Ca2+ free medium 1 h prior to re-addition of glucose. Since it takes >20 min to fully unload Ca2+ stores in permeabilized cells in a Ca2+ -free medium supplemented with EGTA, we speculate that the TECC response in the absence of external Ca2+ reflects release of remaining Ca2+ in organelles. The finding that the steady-state cytosolic Ca2+ level obtained at the end of the TECC response is exactly the same whether or not external Ca2+ is present strongly suggests that steady-state cytosolic Ca2+ levels under these conditions are set by cytosolic pH. Although the vacuole is the main intracellular Ca2+ store in yeast cells and functionally equivalent to Ca2+ transport to the extracellular medium in other eukaryotic cells, active Ca2+ transport mechanisms including Pmr1, Cod1 and Gdt1 are also expressed in the ER/Golgi. In sharp contrast to mutants lacking vacuolar transporters (Pmc1 and/or Vcx1), Ca2+ stored during a Ca2+ pulse was dramatically increased in cells lacking ER/Golgi transporters, with the exception of gdt1. The greatest increase was seen in pmr1 cells. It has previously been reported that pmr1 and cod1 cells display increased Ca2+ influx [18,54,55] in conjunction with calcineurin-induced upregulation of Pmc1 [18,34,54]. In most cell types, depletion of intracellular Ca2+ stores signals the activation of capacitative Ca2+ entry (CEE), occurring through store-operated Ca2+ channels (reviewed in [56]). A CEE-like mechanism involving Cch1/Mid1 activation has also been proposed in yeast cells [18,24]. Therefore, one may argue that the observed increase in Ca2+ pulse release in pmr1 and cod1 cells simply reflects increased Ca2+ influx via Cch1/Mid1 channels which is subsequently sequestered in the vacuole mainly through Pmc1 (Fig. 10). However, there are at least three counter-arguments to this scheme. First, since yeast cells were not exposed to external Ca2+ prior to aeqourin measurements, we expected that calcineurin-induced changes in Pmc1 expression were not largely involved in this effect [57]. This was confirmed in pmr1 cells treated with the calcineurin-inhibiting immunosuppressants CsA or FK506 [58,59]. Second, Ca2+ uptake during the Ca2+ pulse was similarly increased in pmc1/pmr1 and in pmr1/cch1/mid1 cells compared to single Pmr1 deletion indicating that neither Pmc1 nor Cch1/Mid1 channels are per se required for this effect. Finally, it is striking that deletion of Vcx1 abolished the effect in pmr1 cells whereas Pmc1 was required in cod1 cells. Taken together, it seems plausible that upon ER (cod1) or ER/Golgi (pmr1) Ca2+ store depletion, yeast cells activate a mechanism that enhances Ca2+ influx, which in turn stimulates vacuolar Ca2+ uptake or vice versa. However, when vacuolar Ca2+ uptake is suppressed (Pmc1 deletion in cod1 or Vcx1 deletion in pmr1) this mechanism appears to be inactive. It is clear

Please cite this article in press as: P. D’hooge, et al., Ca2+ homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca2+ storage, Cell Calcium (2015), http://dx.doi.org/10.1016/j.ceca.2015.05.004

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Fig. 9. Vacuole morphology and Ca2+ pulse release. Graph showing relationship between Ca2+ pulse release and vacuolar fragmentation in wt and deletion strains. Percentages refer to the number of cells displaying >2 vacuoles. Open black square symbol denotes wt yeast. Filled red, green and black circles denote deletion strains lacking either vacuolar, ER/Golgi or plasma membrane Ca2+ transporters, respectively. Mixed colours denote deletion strains lacking ER/Golgi and vacuolar Ca2+ transporters (green border – red centre and SEM bars) or ER/Golgi and plasma membrane Ca2+ transporters (green border – black centre and SEM bars). The solid line indicates the best-fitting linear function (with r2 = 0.2964 and P = 0.0195). Insets: confocal images of wt and pmr1/pmc1 yeast cells labelled with GFP-tagged Vph1p to visualize vacuolar membranes. Black bar corresponds to 1 ␮m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 10. Ca2+ homeostasis in Saccharomyces cerevisiae. In wt yeast (upper scheme) extracellular Ca2+ enters the cytosol through Cch1/Mid1 channel complex and an unknown transporter (boxed question mark). Resting cytosolic [Ca2+ ] is finely tuned by vacuolar Ca2+ uptake trough Vcx1 and Pmc1 and by Ca2+ uptake in the ER/Golgi trough Pmr1 and Cod1. To account for increased Ca2+ influx in Pmr1 or Cod1 cells a CEE-like mechanism has been proposed (right lower scheme). In response to ER/Golgi Ca2+ depletion plasma membrane Cch1/Mid1 channels become activated (CCE-like mechanism) resulting in increased levels of cytosolic Ca2+ , activation of calcineurin (CN), a CN-induced compensatory increase in expression of Pmc1 and hence increased vacuolar Ca2+ uptake. Based on our work, we suggest that upon ER (cod1) or ER/Golgi (pmr1) Ca2+ store depletion, yeast cells activate a mechanism that enhances Ca2+ influx through not-yet identified plasma membrane Ca2+ transporters (boxed question mark), which in turn stimulates vacuolar Ca2+ uptake and induces vacuolar fragmentation or vice versa. When vacuolar Ca2+ uptake is suppressed (Pmc1 deletion in cod1 or Vcx1 deletion in pmr1) this mechanism appears to be inactive (left lower scheme).

that calcineurin is not involved in this process, however, the molecular identity of the influx route as well as the molecular pathway underlying increased vacuolar Ca2+ uptake in this process remain to be investigated. It is intriguing to note that mutant strains displaying large Ca2+ pulse release also have fragmented vacuolar morphologies. Based on previous studies [38,54,60,61], it is clear that disturbances in ER/Golgi ion homeostasis (Ca2+ and Mn2+ ) may lead to conspicuous defects in protein processing, vesicle trafficking and vacuolar biogenesis. Assuming that proton pomp activity is maintained in both small and large vacuoles, small vacuoles may establish a larger proton gradient owing to their larger surface to volume ratio. Hence, vacuolar Ca2+ sequestration by Vcx1 would be enhanced. Additionally, as a result of vacuolar fragmentation one would expect a significant increase in the amount of free releasable Ca2+ . This can be readily seen as an increase in the fast component of the Ca2+ pulse release in pmr1, pmc1/pmr1 and pmr1/cch1/mid1

strains (Fig. 8). The finding that deletion of Vcx1 in the pmr1 strain reversed both the increase in Ca2+ pulse release and vacuolar fragmentation suggests that the high Ca2+ content in the vacuole is the trigger mechanism that leads to vacuolar fragmentation. In contrast to the pmr1 strain, disruption of Vcx1 did not reverse Ca2+ pulse release in cod1 cells strain while Pmc1 deletion reversed Ca2+ pulse release but not vacuolar fragmentation. This marked difference between cod1 and pmr1 mutants strains may reflect the distinct roles of Pmr1 and Cod1 in adequate supply of Ca2+ or Mn2+ to the Golgi/ER compartment. Future studies are needed to determine how Cod1 deletions may stimulate Pmc1-dependent vacuolar Ca2+ uptake. In contrast to single deletion of Pmr1 or Cod1, Ca2+ pulse release was slightly attenuated in gdt1 cells. Furthermore, Ca2+ responses in cod1/gdt1 were similar to those observed in the single mutant cod1. In agreement with previous results [35], these results suggest that Gdt1 plays only a minor role in ER/Golgi Ca2+ supply.

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In line with previous reports [34,38,39], we also observed that peak TECC responses were significantly enhanced in pmr1 or cod1 cells. The trivial explanation that this effect is merely a consequence of decreased ER/Golgi Ca2+ sequestration can be ruled out as TECC release signals are also increased in these mutants. The changes in peak TECC response and TECC release in the various ER/Golgi deletion strains including pmc1/cod1, pmc1/pmr1, pmr1, cod1, gdt1, cod1/gdt1, pmr1/cch1/mid1 follow a similar pattern as for the Ca2+ pulse release signal. Therefore, we suggest that in addition to reduced ER/Golgi Ca2+ sequestration, increased vacuolar Ca2+ storage in conjunction with increased Ca2+ influx, underlies the larger TECC responses in pmr1 or cod1 cells. TECC responses in vcx1/pmr1 and vcx1/cod1 mainly reflect consequences of Vcx1 deletion and support the notion that Vcx1 plays a dominant role in TECC responses. However, not fitting this picture is the double deletion strain pmr1/cod1. Despite increased Ca2+ pulse release, TECC responses are not affected in this strain. Whether this reflects additional changes in expression or activity of other proteins involved in the TECC response, remains to be investigated. In line with some previous reports [25], we found that TECC responses were not significantly affected in cells lacking both Mid1 and Cch1, two subunits of a high affinity-low capacity plasma membrane Ca2+ channel. TECC responses were also not affected in Mid1 or Cch1 single deletion strains. Therefore, other still unknown plasma membrane Ca2+ transporters should be involved in the TECC response. Possible candidates, although not yet identified at the molecular level, include a low affinity, high capacity Ca2+ channel involved in pheromone-signalling [4], 2 transporters referred as X and M involved in Ca2+ influx in non-stimulated yeast cells grown under standard conditions [13] and the glucose induced Ca2+ influx or GIC pathway [25]. Notably, other studies have shown that TECC responses are abolished in Cch1/Mid1 deletion strains but not in single deletion strains lacking either Cch1 or Mid1 [4,11] suggesting that the Cch1/Mid1 complex is a major Ca2+ influx pathway in yeast cells. Although these differences cannot presently be fully accounted for, a plausible explanation may be related to the composition of the medium used to grow yeast. It is well known that growth culture conditions may strongly affect expression levels of a large number of genes including those involved in ion homeostasis [4,25,62]. For selection purposes and bioluminescence assays, all yeast strains in our study were grown in SD lacking Leucine and Uracil. In previous studies cells were grown in either rich media or minimal media lacking only Uracil [11,25]. Therefore, it is possible that the expression or functionality of Mid1 or Cch1 is modulated by growth culture conditions. In conclusion, we showed that deletion of Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca2+ homeostasis, is associated with abnormal vacuolar morphology, increased vacuolar Ca2+ storage and Ca2+ influx through a mechanism independent of Cch1/Mid1 channels. In contrast, yeast strains lacking Vcx1 and/or Pmc1, two Ca2+ transporters linked to vacuolar Ca2+ homeostasis, exhibited decreased Ca2+ storage. Our analysis further revealed the distinct roles of Vcx1 and Pmc1 in vacuolar Ca2+ uptake. Since yeast cells employ a similar toolkit for Ca2+ handling as that used in animal cells [13,14,63], our findings may be extrapolated to mammalian Ca2+ homeostasis and may help to understand Ca2+ dyshomeostasis associated with human diseases [47,64–74].

Author contribution G.C. and J.W. supervised the project. P.D., C.C., V.V.E., L.F., R.G. and L.M. performed experiments and analyzed data. G.C. wrote the main paper. All authors discussed the results and implications and commented on the manuscript at all stages.

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Conflict of interest statement All co-authors have seen and agreed with the contents of the manuscript, and none of the co-authors has any financial interests to disclose. Acknowledgements This work was supported by a grant from the Research Foundation – Flanders (F.W.O.) to J.W. and G.C., and the Hercules funding (Equipment renewal Flemish government) to G.C. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ceca.2015.05.004 References [1] H. Iida, S. Sakaguchi, Y. Yagawa, Y. Anraku, Cell cycle control by Ca2+ in Saccharomyces cerevisiae, J. Biol. Chem. 265 (1990) 21216–21222. [2] A.F. Batiza, T. Schulz, P.H. Masson, Yeast respond to hypotonic shock with a calcium pulse, J. Biol. Chem. 271 (1996) 23357–23362. [3] A.D. Hartley, S. Bogaerts, S. Garrett, cAMP inhibits bud growth in a yeast strain compromised for Ca2+ influx into the Golgi, Mol. Gen. Genet. 251 (1996) 556–564. [4] E.M. Muller, E.G. Locke, K.W. Cunningham, Differential regulation of two Ca(2+) influx systems by pheromone signaling in Saccharomyces cerevisiae, Genetics 159 (2001) 1527–1538. [5] R. Serrano, A. Ruiz, D. Bernal, J.R. Chambers, J. Arino, The transcriptional response to alkaline pH in Saccharomyces cerevisiae: evidence for calcium-mediated signalling, Mol. Microbiol. 46 (2002) 1319–1333. [6] P.R. Kraus, J. Heitman, Coping with stress: calmodulin and calcineurin in model and pathogenic fungi, Biochem. Biophys. Res. Commun. 311 (2003) 1151–1157. [7] E.M. Muller, N.A. Mackin, S.E. Erdman, K.W. Cunningham, Fig. 1p facilitates Ca2+ influx and cell fusion during mating of Saccharomyces cerevisiae, J. Biol. Chem. 278 (2003) 38461–38469. [8] N. Rispail, D.M. Soanes, C. Ant, R. Czajkowski, A. Grunler, R. Huguet, E. Perez-Nadales, A. Poli, E. Sartorel, V. Valiante, M. Yang, R. Beffa, A.A. Brakhage, N.A. Gow, R. Kahmann, M.H. Lebrun, H. Lenasi, J. Perez-Martin, N.J. Talbot, J. Wendland, A. Di Pietro, Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi, Fungal Genet. Biol. 46 (2009) 287–298. [9] Y. Eilam, M. Othman, D. Halachmi, Transient increase in Ca2+ influx in Saccharomyces cerevisiae in response to glucose: effects of intracellular acidification and cAMP levels, J. Gen. Microbiol. 136 (1990) 2537–2543. [10] J. Nakajima-Shimada, H. Iida, F.I. Tsuji, Y. Anraku, Monitoring of intracellular calcium in Saccharomyces cerevisiae with an apoaequorin cDNA expression system, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 6878–6882. [11] M. Tokes-Fuzesi, D.M. Bedwell, I. Repa, K. Sipos, B. Sumegi, A. Rab, A. Miseta, Hexose phosphorylation and the putative calcium channel component Mid1p are required for the hexose-induced transient elevation of cytosolic calcium response in Saccharomyces cerevisiae, Mol. Microbiol. 44 (2002) 1299–1308. [12] R. Kellermayer, R. Szigeti, M. Kellermayer, A. Miseta, The intracellular dissipation of cytosolic calcium following glucose re-addition to carbohydrate depleted Saccharomyces cerevisiae, FEBS Lett. 571 (2004) 55–60. [13] J. Cui, J.A. Kaandorp, O.O. Ositelu, V. Beaudry, A. Knight, Y.F. Nanfack, K.W. Cunningham, Simulating calcium influx and free calcium concentrations in yeast, Cell Calcium 45 (2009) 123–132. [14] J. Cui, J.A. Kaandorp, P.M. Sloot, C.M. Lloyd, M.V. Filatov, Calcium homeostasis and signaling in yeast cells and cardiac myocytes, FEMS Yeast Res. 9 (2009) 1137–1147. [15] M.S. Cyert, C.C. Philpott, Regulation of cation balance in Saccharomyces cerevisiae, Genetics 193 (2013) 677–713. [16] H. Iida, H. Nakamura, T. Ono, M.S. Okumura, Y. Anraku, MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating, Mol. Cell. Biol. 14 (1994) 8259–8271. [17] M. Fischer, N. Schnell, J. Chattaway, P. Davies, G. Dixon, D. Sanders, The Saccharomyces cerevisiae CCH1 gene is involved in calcium influx and mating, FEBS Lett. 419 (1997) 259–262. [18] E.G. Locke, M. Bonilla, L. Liang, Y. Takita, K.W. Cunningham, A homolog of voltage-gated Ca(2+) channels stimulated by depletion of secretory Ca(2+) in yeast, Mol. Cell. Biol. 20 (2000) 6686–6694. [19] M. Bonilla, K.W. Cunningham, Calcium release and influx in yeast: TRPC and VGCC rule another kingdom, Sci. STKE 2002 (2002) pe17. [20] H. Yoshimura, T. Tada, H. Iida, Subcellular localization and oligomeric structure of the yeast putative stretch-activated Ca2+ channel component Mid1, Exp. Cell Res. 293 (2004) 185–195.

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Please cite this article in press as: P. D’hooge, et al., Ca2+ homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca2+ storage, Cell Calcium (2015), http://dx.doi.org/10.1016/j.ceca.2015.05.004