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¹ Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation, Emilia Romagna Laboratory for Genomics and Biotechnology, University of Ferrara, Ferrara 44100, Italy
² Endocrinology and Reproduction Research Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
³ Department of Physiology, Semmelweis University, Faculty of Medicine, Budapest, 1081 Hungary
⁴ Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Pasteur 3, Warsaw, 02-093, Poland

Correspondence to Rosario Rizzuto: rzr{at}unife.it

Top Abstract Introduction Results Discussion Materials and methods References

 
The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane mediates metabolic flow, Ca²⁺, and cell death signaling between the endoplasmic reticulum (ER) and mitochondrial networks. We demonstrate that VDAC1 is physically linked to the endoplasmic reticulum Ca²⁺-release channel inositol 1,4,5-trisphosphate receptor (IP₃R) through the molecular chaperone glucose-regulated protein 75 (grp75). Functional interaction between the channels was shown by the recombinant expression of the ligand-binding domain of the IP₃R on the ER or mitochondrial surface, which directly enhanced Ca²⁺ accumulation in mitochondria. Knockdown of grp75 abolished the stimulatory effect, highlighting chaperone-mediated conformational coupling between the IP₃R and the mitochondrial Ca²⁺ uptake machinery. Because organelle Ca²⁺ homeostasis influences fundamentally cellular functions and death signaling, the central location of grp75 may represent an important control point of cell fate and pathogenesis.

Abbreviations used in this paper: grp, glucose-regulated protein; IP₃, inositol 1,4,5-trisphosphate; IP₃R, IP₃ receptor; KRB, Krebs-Ringer bicarbonate; MAM, mitochondria-associated membrane; OMM, outer mitochondrial membrane; SERCA, sarcoplasmic reticulum Ca²⁺ ATPase; tBHQ, tert-butyl-benzohydroquinone; VDAC, voltage-dependent anion channel.

	Introduction

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Mitochondria and ER of eukaryotic cells form two intertwined endomembrane networks, and their dynamic interaction controls metabolic flow, protein transport, intracellular Ca²⁺ signaling, and cell death (Ferri and Kroemer, 2001; Berridge et al., 2003; Szabadkai and Rizzuto, 2004; Yi et al., 2004; Brough et al., 2005; Levine and Rabouille, 2005). Mitochondrial Ca²⁺ uptake, via a yet to be identified Ca²⁺ channel of the inner mitochondrial membrane (the mitochondrial Ca²⁺ uniporter), regulates processes as diverse as aerobic metabolism (Hajnoczky et al., 1995), release of caspase cofactors (Pinton et al., 2001), and feedback control of neighboring ER or plasma membrane Ca²⁺ channels (Hajnoczky et al., 1999; Gilabert and Parekh, 2000). A corollary of the efficient mitochondrial Ca²⁺ uptake during IP₃-induced Ca²⁺ release is the close apposition of ER and outer mitochondrial membranes (OMM; Mannella et al., 1998; Rizzuto et al., 1998b; Marsh et al., 2001; Frey et al., 2002). The molecular determinants of this crosstalk, however, are still largely unknown (Walter and Hajnoczky, 2005). Recently, PACS2, which is an ER-associated vesicular-sorting protein, was proposed to link the ER to mitochondria (Simmen et al., 2005). The knockdown of PACS2 led to stress-mediated uncoupling of the organelles, which was also reflected by the inhibition of Ca²⁺ signal transmission.

On the other side, the abundant OMM channel voltage-dependent anion channel 1 (VDAC1) was also suggested to participate in the interaction. It was shown to be present at ER–mitochondrial contacts and to mediate Ca²⁺ channeling to the intermembrane space from the high [Ca²⁺] microdomain generated by the opening of the inositol 1,4,5-trisphosphate receptor (IP₃R; Gincel et al., 2001; Rapizzi et al., 2002). In addition, VDAC1 mediates metabolic flow through the OMM, forming an ATP microdomain close to the ER and sarcoplasmic reticulum Ca²⁺ ATPases (SERCAs; Ventura-Clapier et al., 2004; Vendelin et al., 2004), and both VDAC1 and VDAC2 take part in metabolic and apoptotic protein complexes (Cheng et al., 2003; Colombini, 2004; Lemasters and Holmuhamedov, 2006).

The transfer and assembly of components of cellular protein complexes were shown to be assisted by molecular chaperones, adding a novel function to their role in nascent protein folding (Young et al., 2003; Soti et al., 2005). Accordingly, Ca²⁺ binding–, heat shock–, and glucose-regulated chaperone family members are abundantly present along the Ca²⁺ transfer axis, linking the ER and mitochondrial networks. Well known examples are the Ca²⁺-binding chaperones of the ER lumen (Michalak et al., 2002), immunophilins interacting with ER Ca²⁺-release channels and the mitochondrial permeability transition pore (Bultynck et al., 2001; Forte and Bernardi, 2005), and several heat shock family members localized at the mitochondrial membranes, which are proposed to interact with the components of the mitochondrial permeability transition pore, such as VDAC (He and Lemasters, 2003; Gupta and Knowlton, 2005; Wadhwa et al., 2005). Still, their exact role at the ER–mitochondria interface is not well known, although recently, weak links between chaperones were proposed to stabilize signaling and organellar cellular networks (Csermely, 2004; Soti et al., 2005).

Considering the central position of VDAC at the ER–mitochondrial interface outlined in the previous paragraphs, we used VDAC1 as a starting point for protein biochemical studies, to explore molecular interactions between the ER and mitochondrial networks. We found that through the OMM-associated fraction of the glucose-regulated protein 75 (grp75) chaperone (Zahedi et al., 2006), VDAC1 interacts with the ER Ca²⁺-release channel IP₃R. Organellar Ca²⁺ measurements, using targeted recombinant Ca²⁺ probes, confirmed functional interaction between the IP₃R and the mitochondrial Ca²⁺ uptake machinery, which was abolished by grp75 knockdown.

	Results

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VDAC1, grp75, and IP₃Rs are present in a macromolecular complex at the ER–mitochondria interface
We performed yeast two-hybrid screens of human liver and kidney LexA-AD–fused libraries, using rat VDAC1-LexA-DNA- BD fusion protein as bait. Among the putative interactors we found cytoskeletal elements, which were previously thought to participate in sorting of VDAC or in mitochondrial dynamics (Schwarzer et al., 2002; Varadi et al., 2004) and a group of chaperone proteins (Table I). To investigate whether the chaperones participate in mediating organelle interactions, we focused our attention on the human heat shock 70 kD protein 9B/grp75 (nt 1,456–2,089 from GenBank/EMBL/DDBJ under accession no. BC000478; aa 471–681). The yeast homologue of grp75 is part of the protein import motor associated with TIM23 in the mitochondrial matrix (Neupert and Brunner, 2002), but it was also found in the cytosol and in OMM-associated high molecular weight protein complexes (Ran et al., 2000; Danial et al., 2003; Zahedi et al., 2006). In addition, two further findings indicated that grp75 may be involved in ER–mitochondria Ca²⁺ transfer: first, its C-terminal domain reduced the voltage dependence and cation selectivity of VDAC1 (Schwarzer et al., 2002), and second, grp75 overexpression was shown to promote cell proliferation and protect against Ca²⁺-mediated cell death (Wadhwa et al., 2002a; Liu et al., 2005).

View larger version (49K): [in this window] [in a new window] [Download PPT slide]   Figure 1. IP3R, VDAC1, and grp75 colocalize on the MAM fraction. (A) Protein components of subcellular fractions prepared from rat liver and HeLa cells revealed by immunoblot analysis. Mito, mitochondria; MAM, light mitochondrial fraction; P, heavy mitochondrial fraction, enriched in matrix components; C, crude mitochondrial fraction before Percoll gradient separation. 10 µg of proteins were loaded on 10% SDS-polyacrylamide gels. The presence of IP3Rs was shown by using a non–isotype-specific monoclonal antibody. VDAC1 and grp75 were both present in the MAM, whereas it was free of contamination from inner membrane (Cox-II) and matrix (MnSOD; C) proteins. Different preparations are separated by the dotted line. Blots are representative of more than five experiments. (B) Blue-native and SDS-PAGE 2D separation of the MAM fraction (below BN) and Mito P proteins (above BN; for preparation of native subcellular fractions, see Materials and methods and A). The native fractions were solubilized and separated on an acrylamide gradient gel in the first dimension. The capillary gel was stacked over a 10% SDS-polyacrylamide gel and separated, and the proteins were immunoblotted against the IP3Rs, grp75, and VDAC1. A typical result of an immunoblot from three separate experiments is shown. (C) The MAM and Mito P fractions (50 µg of proteins) were subjected to proteinase K digestion (50 µg/ml) and the presence of grp75 and MnSOD was revealed by immunoblotting. Hyposmotic shock (50 mM mannitol, 5 mM Hepes, and 0.1 mM EGTA for 30 min at room temperature) was applied to the Mito P fraction to induce release of matrix proteins. (D–F) Coimmunoprecipitation of grp75 with IP3R and VDAC1. Total cellular proteins were used for immunoprecipitation with a polyclonal IP3R1 (D), a polyclonal VDAC (E), and a monoclonal grp75 (F) antibody, and the precipitated protein fractions were separated on 10% SDS-polyacrylamide gels and immunoblotted against IP3Rs, grp75, and VDAC1. The input homogenate fractions, the IgG controls, and the immunoprecipitates are shown.

To further investigate the arrangement of the grp75–VDAC–IP₃R complex, we used coimmunoprecipitation studies of the involved proteins. Immunoprecipitation of both IP₃Rs and VDAC led to the coprecipitation of grp75 (Fig. 1, D and E, respectively), but no IP₃R was found in the VDAC precipitate, and no VDAC was detectable in the IP₃R precipitate. However, immunoprecipitation of grp75 led to the copurification of both VDAC and the IP₃R (Fig. 1 F). These results strongly suggest that grp75 has a central role in setting up the protein complex with VDAC and the IP₃R. Moreover, the interactions were detected both in the presence and absence of Mg²⁺-ATP (unpublished data), further suggesting the scaffolding, rather than chaperoning, function of grp75 in the complex.

Direct regulation of mitochondrial Ca²⁺ uptake by the IP₃R ligand-binding domain
If the IP₃R is in a macromolecular assembly with VDAC, we assumed that the mitochondrial Ca²⁺ uptake machinery might be regulated by the large cytoplasmic domain of the IP₃R. This scheme was also supported by previous studies showing that the ligand-binding domain of the IP₃R (aa 224–605; denoted as IP₃R-LBD_224-605), located on the surface of the cytoplasmic domain, participates in intramolecular interactions with other IP₃R domains (Boehning and Joseph, 2000), as well as in linking the receptor with other protein partners (Bosanac et al., 2004). To assess a direct role of the IP₃R in mitochondrial Ca²⁺ uptake, we coexpressed in HeLa cells mRFP1-tagged IP₃R-LBD_224-605 with cytosolic (cytAEQ) or mitochondrially targeted (mtAEQmut) aequorin-based Ca²⁺ probes, and evaluated global and organellar Ca²⁺ responses to agonist stimulation. After reconstitution with the aequorin cofactor coelenterazine, cells were challenged with histamine (in incremental doses from 1 to 100 µM), and luminescence was measured and converted to [Ca²⁺]. Recombinant expression of the IP₃R-LBD_224-605 caused a marked increase in mitochondrial Ca²⁺ uptake at each agonist concentration applied, in spite of reduced cytoplasmic Ca²⁺ response ([Ca²⁺]_c), because of IP₃ buffering and consequent reduction of IP₃-induced Ca²⁺ release from the ER (see Fig. 2 [A and B] and Fig. S2 [available at http://www.jcb.org/cgi/content/full/jcb.200608073/DC1] for the lower agonist concentrations). The effect of the IP₃R-LBD_224-605 was presumably exerted on the OMM because targeting the IP₃R-LBD_224-605 to the OMM surface (by fusing to an N-terminal AKAP1 domain) augmented its stimulatory effect (see Fig. 3 A for intracellular localization of the mRFP1-tagged construct and Fig. 2 B for the effect on [Ca²⁺]_m). Morphological imaging and mitochondrial loading with the potential-sensitive dye teramethylrhodamine methyl ester showed that the effect was not caused by changes in mitochondrial morphology (Fig. 3 A) or to the modification of mitochondrial membrane potential (not depicted).

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Effect of the IP3R ligand-binding domain on mitochondrial Ca2+ uptake. (A and C) HeLa cells were transfected with mitochondrially targeted (mtAEQmut; top) and cytosolic aequorin (bottom). Control traces are shown in black; traces from cells cotransfected with the IP3R-LBD224-605 (A) and the IP3R-LBD224-605 K508 mutant (C) are shown in gray. Traces are representative of >15 experiments from >5 preparations. (B) Effect of the cytosolic-, OMM-, and ER-targeted IP3R-LBD224-605 on peak mitochondrial and cytosolic Ca2+ responses (top and bottom, respectively). (D) Effect of the OMM-IP3R-LBD224-605 (K508A), the IP3-binding PH domain of the p130 PLC-like protein (OMM-p130-PH), and the OMM targeted N-terminal (1-604 aa) part of the IP3R (OMM-IP3R-LBD1-604), on mitochondrial (top) and cytoplasmic Ca2+ responses (bottom) after 100 µM histamine stimulation. Data in B and D were normalized to mean of the control group. Mean ± SEM of variation is shown as percentage. Cells were transfected, and [Ca2+] was measured as described in Materials and methods. Values are shown. *, P < 0.05; **, P < 0.01. For absolute values see Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200608073/DC1.

View larger version (59K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Intracellular localization of OMM- and ER-targeted IP3R-LBD224-605. Cells were transfected with OMM-IP3R-LBD224-605-mRFP1 (A) or ER-IP3R-LBD224-605-mRFP1 (B) and loaded with the mitochondrial dye MitoTracker Green. Images on the left show mitochondrial structure, middle images show images of IP3R-LBD224-605-mRFP1 fluorescence, and images on the right show colocalization of the green and red signals. Insets show magnified images of the mitochondrial and ER networks. Bars: (A and B) 10 µm; (insets) 2 µm.

Stimulation of mitochondrial Ca²⁺ uptake by the IP₃R-LBD is the result of specific protein interactions at the ER–OMM interface
Based on our conclusions, we further investigated whether the effect of the N-terminal cytosolic domain of the IP₃R reflects specific protein–protein interactions at the ER–mitochondrial contacts. We first verified that the effect of IP₃R-LBD_224-605 on mitochondrial Ca²⁺ uptake is independent of IP₃ buffering. For this purpose, we used a point-mutated (K508A) IP₃R-LBD_224-605, which is unable to bind IP₃. The K508 mutant increased the [Ca²⁺]_m rise in a manner similar to the wild-type (although slightly less efficient), but, as expected, did not modify the [Ca²⁺]_c response (Fig. 2, C and D). The capacity of an IP₃-insensitive IP₃R-LBD_224-605 to enhance mitochondrial Ca²⁺ uptake was also confirmed in digitonin-permeabilized HeLa cells. In this case, mitochondrial Ca²⁺ uptake is exclusively dictated by the perfused [Ca²⁺], and it is totally independent of IP₃R activity. In permeabilized cells, Ca²⁺ uptake was triggered by the perfusion of an intracellular buffer containing Ca²⁺ buffered at 1 µM. Under those conditions, in which protein interactions might have been affected by the application of digitonin, both the wild-type and the K508A OMM-IP₃R-LBD_224-605 increased the rate of mitochondrial Ca²⁺ uptake, although also in this case the wild-type was more efficient (14.71 ± 4.66% increase, n = 25, P < 0.01 vs. 6.58 ± 4.23% increase, n = 25, P > 0.05).

The notion that IP₃ binding cannot account for the mitochondrial effect was further confirmed by the demonstration that a structurally unrelated IP₃-binding protein domain, the PH domain of the PLC-like protein p130 (p130PH; Lin et al., 2005), targeted to the OMM, reduced both the [Ca²⁺]_m and [Ca²⁺]_c responses (Fig. 2 D). Interestingly, the reduction of the [Ca²⁺]_m response was more pronounced for the OMM-targeted PH domain than for the untargeted cytosolic version of the IP₃ buffer, although the two proteins were equally effective on [Ca²⁺]_c. These data (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200608073/DC1) further stress the strict dependence of mitochondrial Ca²⁺ homeostasis on the ER– mitochondrial contacts, and thus on the Ca²⁺ release occurring in these microdomains.

The IP₃R-LBD_224-605 was also shown to play an important role in the regulation of IP₃R channel activity by interacting with the N-terminal repressor domain (aa 1–223; Boehning and Joseph, 2000; Varnai et al., 2005). Still, expressing the entire N-terminal surface domain of the IP₃R, targeted to the exterior of the OMM (OMM-IP₃R_1-604), augmented mitochondrial Ca²⁺ uptake (Fig. 2 D). These results exclude that the stimulatory effect of the IP₃R-LBD_224-605 was exerted through unmasking this intramolecular interaction in the endogenous IP₃R; instead, they support a model in which the entire N-terminal IP₃R exerts direct activation on the mitochondrial Ca²⁺ uptake machinery.

Finally, we investigated the regulatory activity on mitochondria of the IP₃R-LBD_224-605 when the [Ca²⁺]_c rise is elicited in the cell by the opening of plasma membrane channels. Under those conditions, not only the [Ca²⁺]_c rise is IP₃R-independent, but the [Ca²⁺]_c and ensuing [Ca²⁺]_m increases are markedly slower and smaller than upon ER Ca²⁺ release. We thus measured [Ca²⁺]_m after emptying the ER Ca²⁺ pool with the SERCA blocker tert-butyl-benzohydroquinone (tBHQ) in Ca²⁺-free medium and re-adding CaCl₂. This protocol induces capacitative Ca²⁺ entry, causing a [Ca²⁺]_c rise and subsequent mitochondrial Ca²⁺ uptake. As presented in Fig. 4, IP₃R-LBD_224-605–expressing cells showed an 60% increase in the influx-dependent [Ca²⁺]_m response (top), even if the [Ca²⁺]_c rise remained unaltered (bottom). This increase in [Ca²⁺]_m was almost doubled, as compared with the effect after histamine-/IP₃-induced Ca²⁺ release from the ER (Fig. 2 B). Thus, we concluded that local IP₃ buffering masks the stimulatory effect of the IP₃R-LBD_224-605 upon ER Ca²⁺ release, and, indeed, the effect of the IP₃R-LBD is established at the ER–mitochondrial contacts.

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Figure 4. The effect of IP3R-LBD224-605 on mitochondrial Ca2+ uptake after capacitative Ca2+ influx. [Ca2+]m and [Ca2+]c (top and bottom, respectively, in A and B) were measured in HeLa cells and transfected with mtAEQmut and cytAEQ, respectively. After ER depletion in Ca2+-free medium (100 µM KRB-EGTA; 4 min), Ca2+ influx was induced by the re-addition of 2 mM CaCl2 to the extracellular medium. (A) Representative traces of control (black traces) and OMM-IP3R-LBD224-605–cotransfected cells (gray traces) are shown. ([Ca2+]m peak in controls, 12.1 ± 2.11 µM; [Ca2+]m peak in OMM-IP3R-LBD224-605–expressing cells, 21.2 ± 4.00 µM; P = 0.05; [Ca2+]c peak in controls, 0.96 ± 0.04 µM; [Ca2+]c peak in OMM-IP3R-LBD224-605–expressing cells, 1.04 ± 0.03 µM). In B, data normalized to the mean ± the SEM of the control group are shown as percentages. For absolute values see Table S1. **, P < 0.01.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Coupling of the ER and mitochondrial Ca2+ channels depends on the presence of grp75. Mitochondrial Ca2+ uptake was measured in control siRNA–transfected HeLa cells (control); after siRNA-driven down-regulation of grp75 (siRNA-grp75); control siRNA and OMM-IP3R-LBD224-605–transfected cells; and siRNA-grp75 and OMM-IP3R-LBD224-605 cotransfected cells. Cells were also cotransfected with the mtAEQmut probe and mitochondrial Ca2+ response to 100 µM histamine was measured as described in the Materials and methods. Inset shows the effect of grp75 siRNA on grp75 levels after 24 h of transfection. Controls transfected only with Lipofectamine showed no difference in respect to control siRNA (not depicted). (B) Silencing of grp75 reverts the stimulatory effect of IP3R-LBD224-605 targeted both to the OMM and ER surface. The percent increase of [Ca2+]m peaks normalized to the mean of controls are shown in cells cotransfected with mtAEQmut and control siRNA (siRNA-grp75) and OMM-IP3R-LBD224-605 or ER-IP3R-LBD224-605 after stimulation with 100 µM histamine. The stimulatory effect of both the OMM- and ER-targeted IP3R-LBD224-605 was inhibited after the cotransfection with siRNA-grp75 (+), whereas the control Ca2+ peaks remained unaffected. Data normalized to the mean ± the SEM of the control group are shown in percentages. For absolute values see Table S1. *, P < 0.05; **, P < 0.01.

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Effect of grp75 overexpression on mitochondrial Ca2+ responses and steady-state [Ca2+]er. (A and B) HeLa cells were cotransfected with mtAEQmut or erAEQmut probes (controls) and mouse grp75. [Ca2+]m was measured as described in Fig. 2, after stimulation with 100 µM histamine, as indicated in A. The percent variation (± SEM) of [Ca2+]m peaks normalized to the mean of controls are shown in B (left); the effect of grp75 on steady-state [Ca2+]er is shown on the right. Steady-state [Ca2+]er was measured after refilling of the ER in the presence of 1 mM CaCl2 in the extracellular medium (n = 10, from four separate experiments). Before measurements, erAEQmut-transfected cells were reconstituted with coelenterazine n, after ER Ca2+ depletion in a solution containing 0 [Ca2+], 600 µM EGTA, and 1 µM ionomycin, as previously described (Chiesa et al., 2001). For [Ca2+]m values see Table S1. [Ca2+]er in controls, 416 ± 19.3 µM; in grp75-overexpressing cells: 334 ± 13.6 µM; P < 0.05. *, P < 0.05. (C and D) [Ca2+]m (top) and [Ca2+]c (bottom) were measured in control and grp75cyt-expressing cells, after induction of capacitative Ca2+ influx, after ER depletion with tBHQ in Ca2+-free medium (100 µM KRB-EGTA; 4 min) and readdition of 2 mM CaCl2. Representative traces of controls, cells cotransfected with OMM-IP3R-LBD224-605, grp75cyt, or both are shown. The percent increase (± SEM) of [Ca2+]m peaks normalized to the mean of controls are shown in D. [Ca2+]m peak in controls, 9.7 ± 1.2 µM; in OMM-IP3R-LBD224-605–expressing cells, 13.0 ± 1.8 µM; grp75cyt-expressing cells, 13.2 ± 1.6 µM; OMM-IP3R-LBD224-605 /grp75cyt–expressing cells, 18.8 ± 2.48. *, P < 0.05; **, P < 0.01.

	Discussion

Top Abstract Introduction Results Discussion Materials and methods References

How is the newly identified regulatory activity on mitochondrial Ca²⁺ uptake exerted? In principle, two different mechanisms can be envisioned. In the first, grp75 could be involved in scaffolding the ER–mitochondria contacts, and thus determines the number of sites in which mitochondria are exposed to the high [Ca²⁺] microdomains generated at the mouth of IP₃Rs. Fluorescent labeling studies of the ER and mitochondria revealed a partial (5–20%) colocalization, reflecting these interactions. However, no increase in colocalization has been observed by overexpression of grp75 (or of the IP₃R-LBD_224-605; unpublished data), suggesting that they do not directly function as structural determinants of the contacts. In a second scenario, grp75 could control the interaction of ER and mitochondrial proteins at the existing organelle contacts, and thus allow cross-talk between signaling partners, e.g., the ion channels of the two membranes. Indeed, grp75, as shown by its knockdown and overexpression models, was necessary and sufficient for the stimulatory effect of the IP₃R-LBD_224-605 on mitochondrial Ca²⁺ uptake. Moreover, the proteomic data also highlight the central role of grp75 in this interaction. VDAC and IP₃Rs coprecipitate with grp75, and the chaperone is coimmunoprecipitated by both anti-IP₃R and -VDAC antibodies, indicating that it is the key assembling molecule in the loose interaction between the two ion channels.

Within the IP₃R–grp75–VDAC complex, potentiation of mitochondrial Ca²⁺ accumulation by the IP₃R-LBD_224-605 does not require IP₃ binding, as demonstrated by the fact that it is retained by the K508A mutant, which is unable to bind IP₃ (Varnai et al., 2005). Although the mutant shows the same stimulatory effect (Fig. 2), one should remember that wild-type IP₃R-LBD_224-605, because of IP₃ buffering, reduces ER Ca²⁺ release, and thus conclude that the wild type is somewhat more effective than the mutant. To further confirm independence from IP₃ buffering, we measured mitochondrial Ca²⁺ uptake after capacitative influx through the plasma membrane (Figs. 4 and 6). Also, under those experimental conditions, the IP₃R-LBD_224-605 potently stimulated mitochondrial Ca²⁺ uptake.

As for the molecular mechanism of the effect on the mitochondrial Ca²⁺ machinery, different scenarios could be envisioned. In the first, the recombinantly expressed IP₃R-LBD, both from the OMM and ER side, could interact with the endogenous IP₃R itself, and modify the probability of its interaction with grp75/VDAC. Indeed, it was previously shown that intramolecular interactions between different domains of the IP₃R, such as the 224–605 minimal IP₃-binding domain and the 1–223 N-terminal repression domain, regulate IP₃R channel opening upon IP₃ binding. Thus, one could hypothesize that the high expression levels of IP₃R-LBD_224-605 represses an interaction between the extreme N-terminal of the endogenous receptor and grp75/VDAC. To clarify this issue, we expressed the whole (aa 1–604) IP₃R-LBD, which is targeted to the OMM. The IP₃R-LBD_1-604 had the same effect as IP₃R-LBD_224-605, thus, excluding competition of these two cytoplasmic, N-terminal domains of the IP₃R. In the second, simpler scenario, the IP₃R-LBD_224-605 mimics the effect of the endogenous IP₃R. Thus, it directly enhances mitochondrial Ca²⁺ uptake by maximizing, within the macromolecular complex, the interaction with the mitochondrial VDAC channel. Indeed, the density of the exogenous IP₃R-LBD_224-605, based on fluorescence labeling (Varnai et al., 2005) and Scatchard plot analysis of IP₃ binding (Wibo and Godfraind, 1994), can be assumed to be at least one order of magnitude higher than the endogenous receptor, and indeed, high expression levels were necessary for the effect of IP₃R on mitochondrial Ca²⁺ uptake.

The central role of grp75 in the IP₃R-LBD–induced augmentation of Ca²⁺ uptake was clearly shown by the siRNA-driven silencing of the protein, leading to the abolition of the effect. Conversely, high-level expression of grp75 induced a compound effect involving at least three different locations, as follows: (1) the ER, decreasing the steady [Ca²⁺]_er level; (2) the OMM, interacting with VDAC, whose permeability/ion selectivity was shown to be modified by grp-75 binding (Schwarzer et al., 2002); and (3) the mitochondrial matrix, modifying mitochondrial parameters, such as pH or Ca²⁺ buffering capacity. Expression of the cytosolic grp75 and measurement of Ca²⁺ influx–induced mitochondrial Ca²⁺ uptake allowed us to eliminate the intramitochondrial effect and changes of ER Ca²⁺ handling. Importantly, mitochondrial Ca²⁺ uptake in this approach was markedly increased, and grp75_cyt potentiated the effect of OMM-IP₃R-LBD, clarifying the effect of the OMM-associated pool of grp75.

In conclusion, we demonstrated that the IP₃R is part of a signaling complex that directly controls Ca²⁺ uptake into mitochondria. Much remains to be understood, but by these results the concept of macromolecular assembly of signaling elements, previously put forward for several plasma membrane channels, can be extended to defined microdomains at the ER–mitochondrial interface. Such an arrangement highlights novel routes for pharmacological intervention that may be used for the modulation of downstream events such as metabolism and apoptosis.

	Materials and methods

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Yeast two-hybrid screening
Yeast two-hybrid screening was carried out using the pLexA system according to the protocol of Gyuris et al. (1993). For details see Supplemental materials and methods (available at http://www.jcb.org/cgi/content/full/jcb.200608073/DC1).

Subcellular fractionation and proteomic analysis
HeLa cells and rat liver were homogenized, and crude mitochondrial fraction (8,000-g pellet) was subjected to separation on a 30% self-generated Percoll gradient, as previously described (Vance, 1990). A low-density band (denoted as the MAM fraction) and a high-density band (denoted as Mito P) were collected and analyzed by immunoblotting and Blue native/SDS-PAGE 2D separation, which are described in detail in the Supplemental materials and methods. Proteinase K (Sigma-Aldrich) digestion was performed with 50 µg enzyme in the presence of 50 µg proteins (10 min, on ice) in solution A used to resuspend subcellular fractions (250 mM mannitol, 5 mM Hepes, and 0.5 mM EGTA, pH 7.4). Hyposmotic shock (50 mM mannitol, 5 mM Hepes, and 0.1 mM EGTA, pH 7.4, for 30 min at room temperature) was applied to induce mitochondrial swelling.

IP₃R and grp75 expression constructs
Mouse grp75, cloned into the expression vector pTOPO (Invitrogen), was provided by R. Wadhwa (University of Tokyo, Tokyo, Japan; Wadhwa et al., 1993). Full-length mouse IP₃R-1 was obtained from K. Mikoshiba (RIKEN Brain Science Institute, Wako City, Saitama, Japan). The constructs encoding the fusion proteins of the PH domain of the p130 protein (from GenBank/EMBL/DDBJ under accession no.D45920; residues 95–233) and the IP₃R-LBD domain (residues 224–605) of the human IP₃R-1 with monomeric red fluorescent protein (mRFP1), GFP, or YFP, as well as the strategies for ER targeting, have been previously described (Lin et al., 2005; Varnai et al., 2005). For OMM tethering, the N-terminal mitochondrial localization sequence of the mouse AKAP1 protein (from GenBank/EMBL/DDBJ under accession no. V84389; residues 34–63) was fused to the N termini of the IP₃R-LBD and p130PH constructs through a short linker (DPTRSR). The OMM-IP3-LBD_1-604-mRFP1 construct was obtained by amplification of the 1–604 fragment of IP₃R-1 cDNA and insertion into the AKAP1/mRFP1 vector. The GRP75cyt cDNA was amplified from a human liver cDNA library (Origene) using the primers 5'-CCCAAGCTTATGAAGGGAGCAGTTGTTGGTATTG-3' and 5'-CGCGGATCCTTACTGTTTTTCCTCCTTTTGATC-3'. After digestion with HindIII and BamHI, the product was ligated into the pcDNA3 plasmid (Invitrogen) digested with the same restriction enzymes. The construct was verified with bidirectional sequencing.

Transient transfection was done by the Ca²⁺-phosphate precipitation technique. Experiments were performed 24–36 h after transfection.

Dynamic in vivo [Ca²⁺] measurements with targeted aequorin probes
cytAEQ-, mtAEQmut-, or erAEQmut-expressing cells were reconstituted with coelenterazine and transferred to the perfusion chamber, and light signal was collected in a purpose-built luminometer and calibrated into [Ca²⁺] values, as previously described (Chiesa et al., 2001). All aequorin measurements were performed in Krebs-Ringer bicarbonate (KRB) containing 1 mM CaCl₂ (KRB/Ca²⁺; Krebs-Ringer modified buffer: 135 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 0.4 mM K₂HPO₄, 1 mM CaCl₂, 5.5 mM glucose, and 20 mM Hepes, pH 7.4). [Ca²⁺]_c after capacitative Ca²⁺ influx was measured by preincubating HeLa cells with the SERCA blocker tBHQ (100 µM) in a KRB solution containing no Ca²⁺ and 100 µM EGTA. Cytoplasmic Ca²⁺ signal and mitochondrial Ca²⁺ uptake were evoked by adding 2 mM CaCl₂ to the medium. For [Ca²⁺]_er measurements, erAEQmut-transfected cells were reconstituted with coelenterazine n, after ER Ca²⁺ depletion in a solution containing 0 [Ca²⁺], 600 µM EGTA, and 1 µM ionomycin, as previously described (Szabadkai et al., 2004). Experiments in permeabilized HeLa cells were performed as previously described (Rapizzi et al., 2002), except that 25 µM digitonin was used to preserve ER–mitochondrial contacts.

Imaging techniques
For 3D morphological image acquisition, the cells were transfected with mRFP1-fused IP₃R-LBD_224-605 constructs and loaded with 50 nM MitoTracker Green (Invitrogen) for 20 min at 37°C. For morphological studies, cells were placed in a thermostatted chamber at 37°C in KRB/Ca²⁺ solution and imaged using an inverted microscope (Axiovert 200M; Carl Zeiss MicroImaging, Inc.) using a 63x/1.4 Plan-Apochromat objective, a CoolSNAP HQ interline charge-coupled device camera (Roper Scientific) and the MetaMorph 5.0 software (Universal Imaging Corp.). Z-series images were deconvolved using the PSF-based Exhaustive Photon Reassignment deconvolution software (Carrington et al., 1995; Rizzuto et al., 1998a), running on a Linux-based PC. For colocalization analysis, thresholded images were 3D rendered using the Data Analysis and Visualization Environment software (Lifshitz, 1998; Rapizzi et al., 2002). To approximate real colocalization, and to exclude artificial ones produced by the noise of the signal, only the voxels with <50% difference in their normalized intensity were taken into account.

Online supplemental material
Table S1 shows [Ca²⁺]_m and [Ca²⁺]_c responses of HeLa cells expressing the constructs in this study. Fig. S1 shows the proteomic analysis of molecular components of the MAM fraction. Fig. S2 shows the effects of IP₃R-LBD_224-605 on cytoplasmic Ca²⁺ responses and ER Ca²⁺ homeostasis. Fig. S3 shows the effect of cytosolic- and OMM-targeted p130-PH domain on mitochondrial Ca²⁺ uptake. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200608073/DC1.

	Acknowledgments

 
We thank Drs. R. Wadhwa and P. Csermely for helpful discussions and M. Negrini and C. Schwienbacher for help with yeast two-hybrid studies.

This work was supported by grants from Telethon-Italy, the Italian Association for Cancer Research, the Italian University Ministry (Programmi di Ricerca di Rilevante Interesse Nazionale, Italian Investment Fund for Basic Research, and local research grants), the Emilia-Romagna Programma Regionale per la Ricerca Industriale, l'Innovazione e il Trasferimento Tecnologico program, the Ferrara Objective 2 funds, and the Italian Space Agency to R. Rizzuto. P. Várnai was supported by the Hungarian Scientific Research fund, the Medical Research Council, and the Hungarian National Committee for Technological Development. M.R. Wieckowski was a recipient of a European Molecular Biology Laboratory short-term fellowship. Part of the work by G. Szabadkai was supported by a Marie-Curie individual fellowship.

Submitted: 11 August 2006

Accepted: 20 November 2006

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