Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca²⁺ channels

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.

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).

Based on these findings, we used further biochemical approaches to investigate the role of grp75 at the ER–mitochondria contact sites. We took advantage of a previously developed method to purify a mitochondria-associated ER subfraction (mitochondria-associated membrane (MAM) fraction [Vance, 1990]). The MAM was previously shown to be enriched in lipid synthases and transferases (Vance, 2003), and it likely represents the membrane microdomain engaged in ER–mitochondrial Ca²⁺ transfer (Filippin et al., 2003; Szabadkai and Rizzuto, 2004; Yi et al., 2004). Indeed, immunoblot screening of the MAM fraction, purified from rat liver and HeLa cells, revealed the presence of grp75, as well as Ca²⁺ channels from both the OMM (VDAC1) and the ER (IP₃R; Fig. 1 A). In liver cells, given the higher yield, the microsomal fraction and the different mitochondrial extracts (the crude mitochondrial pellet [Mito C], the low-density MAM, and the high-density mitochondrial fraction containing the matrix proteins [Mito P]) were separately analyzed. As expected, VDAC and grp75 are not enriched in the MAM, given that the former is highly expressed throughout the OM (because of its role in ion and metabolite diffusion) and the latter is mostly in the matrix (but the two pools show different macromolecular assembles; see following paragraph). Conversely, the IP₃R, besides the microsomes, is present in the crude mitochondria and the MAM fractions and is absent in the purified mitochondria (Fig. 1 A).

We next investigated whether grp75 and the ER and mitochondrial Ca²⁺ channels are part of the macromolecular complex, and whether the grp75 pool involved is that present in the MAM fraction. For this purpose, we separately analyzed the MAM and the Mito P fractions by 2D Blue native (Fig. 1 B; first dimension, central part of the image) and SDS-PAGE protein separation. In the latter dimension, grp75, IP₃R, and VDAC were revealed by immunoblotting. Although VDAC1 was present in different amounts in complexes of a wide molecular weight range, we found a specific complex characterized by the presence of VDAC, the IP₃R, and grp75, suggesting their interaction in the native state. The specificity of the complex formation of VDAC, the IP₃R, and grp75 was corroborated by the finding that SERCA2b showed different localization in the 2D separation (Fig. S1). In Mito P, grp75 was found in lower molecular weight complexes (<400 kD), which is similar to previous data in yeast (Dekker et al., 1997), confirming that grp75 is involved in high molecular weight protein–protein interactions only in the MAM. To confirm the different location of the two grp75 pools, we verified that whereas the matrix-localized grp75 was resistant to proteinase K digestion (similar to the matrix enzyme MnSOD; Fig. 1 C), grp75 of the MAM fraction was degraded by the enzyme, documenting its association with the cytosolic surface of mitochondria.

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.

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 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).

To confirm that activation of mitochondrial Ca²⁺ uptake can be exerted from the original site of the IP₃R (i.e., from the ER membrane), we expressed IP₃R-LBD_224-605 fused to a C-terminal ER-targeting sequence derived from the yeast UBC6 protein (denoted as ER-IP₃R-LBD_224-605; Varnai et al., 2005). Expression of this construct reduced the steady-state ER [Ca²⁺] ([Ca²⁺]_er) and IP₃-induced Ca²⁺ release (Fig. S2 and Fig. 2 B, respectively), which were probably caused by direct activation of the IP₃R, as previously reported for COS-7 cells (Varnai et al., 2005), although store depletion was incomplete in HeLa cells at the expression levels of this study. Still, most importantly, expression of the ER-targeted IP₃R-LBD_224-605 augmented mitochondrial Ca²⁺ accumulation after cellular stimulation by histamine, similar to what was observed upon expression of the OMM-targeted IP₃R-LBD domain (Fig. 3 B shows the intracellular localization of ER-IP₃R-LBD_224-605; Fig. 2 B shows the stimulatory effect of ER-IP₃R-LBD_224-605 on [Ca²⁺]_m). These results strongly suggested that the IP₃R, acting from the ER surface, regulates mitochondrial Ca²⁺ uptake at an OMM site, independent of its Ca²⁺-channeling function.

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) 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.

Because our proteomic studies suggested that the interaction of the VDAC and IP₃R channels is mediated by grp75, we investigated whether the stimulatory effect of the OMM-targeted IP₃R-LBD_224-605 on mitochondrial Ca²⁺ uptake requires the presence of grp75. A first series of experiments showed that strong inhibition of grp75 expression (48 h after transfection) in itself strongly reduced mitochondrial Ca²⁺ uptake, most probably because of alterations of mitochondrial function through inhibition of protein import and Δψ_m loss (unpublished data). Thus, we opted for a lower silencing efficiency by conducting experiments 24 h after transfection (Fig. 5, inset). We expressed control and grp75 siRNAs in HeLa cells, cotransfecting them with the IP₃R-LBD_224-605 construct and mtAEQmut. Under those conditions, grp75 siRNA had no effect on the [Ca²⁺]_m response to histamine stimulation (Fig. 5, A and B). However, the down-regulation of grp75 prevented the stimulatory effect on mitochondrial Ca²⁺ uptake of the IP₃R-LBD_224-605, which was expressed both on the OMM and the ER surface (Fig. 5 B). Thus, we concluded that grp75 is not only physically associated with the IP₃R–VDAC1 complex, but is also necessary for functional coupling between these proteins. These results also show that although moderate knockdown of grp75 does not interfere with its function in the mitochondrial matrix, in accordance with previous results on mitochondrial protein import (Sanjuan Szklarz et al., 2005), the low amount of grp75 at the ER–mitochondrial contacts is a limiting factor for the stimulatory effect of the IP₃R-LBD.

In the final set of experiments, we further investigated the role of grp75 in mitochondrial Ca²⁺ uptake regulation by overexpressing the protein. Most likely caused by its differentially localized pools, grp75 appeared to modify mitochondrial Ca²⁺ uptake after IP₃-induced Ca²⁺ release through diverse mechanisms. Indeed, as shown in Fig. 6 (A and B), overexpression of the wild-type protein led to reduced histamine-induced [Ca²⁺]_m response. However, at the same time, it also significantly decreased the steady-state [Ca²⁺]_er level (Fig. 6 B, right), thus, reducing the driving force for IP₃-induced Ca²⁺ release, which in turn might be responsible for the dampened mitochondrial Ca²⁺ accumulation. This parallel reduction of [Ca²⁺]_er and [Ca²⁺]_m may reflect two different effects of grp75: (1) OMM-localized grp75, presumably through the interaction with the IP₃R or other members of the ER Ca²⁺-handling machinery, may increase the Ca²⁺ leak from the ER through the IP₃R, as previously shown for Bcl-2 (Pinton et al., 2000; Bassik et al., 2004); (2) matrix-localized grp75 may modify mitochondrial parameters (e.g., pH) or import of Ca²⁺-handling proteins, leading to altered mitochondrial Ca²⁺ uptake, as well as the ATP supply for ER Ca²⁺ accumulation through the SERCA pumps. To dissect these effects, we again used the approach of measuring IP₃-independent mitochondrial Ca²⁺uptake after capacitative Ca²⁺ influx. In addition, to distinguish OMM-based effects from those in the mitochondrial matrix, we expressed a truncated grp75 lacking the N-terminal 51-aa mitochondrial-targeting sequence, and thus unable to enter the mitochondrial matrix. Ca²⁺ influx was induced by depleting the ER Ca²⁺ store with tBHQ in the absence of extracellular Ca²⁺, as described in the previous section (Fig. 4). This “cytosolic” form of grp75 (grp75_cyt) did not change the bulk cytosolic [Ca²⁺] response to readdition of Ca²⁺ in the extracellular medium, but significantly increased mitochondrial Ca²⁺ accumulation (Fig. 6, C and D). Moreover, grp75_cyt further potentiated the stimulatory effect of IP₃R-LBD_224-605 (Fig. 6, C and D), confirming the results obtained with siRNA grp75 and proving that the amount of grp75 present at the OMM in the VDAC–grp75–IP₃R complex is a limiting factor of the positive effect of the IP₃R-LBD on mitochondrial Ca²⁺ uptake. Lastly, by coexpressing grp75_cyt and IP₃R-LBD_224-605, we achieved a very high stimulation of mitochondrial Ca²⁺ uptake rate during capacitative Ca²⁺influx (i.e., upon the increase of bulk [Ca²⁺]_c to ∼1 μM). Thus, we concluded that the VDAC–grp75–IP₃R complex renders mitochondria more sensitive at low extramitochondrial [Ca²⁺], as compared with higher local [Ca²⁺]_c increases during IP₃-induced Ca²⁺ release (compare the effect of IP₃R-LBD_224-605 on [Ca²⁺]_m in Fig. 2 and Fig. 4 or Fig. 6). Indeed, by overexpression of grp75_cyt we could not observe a significant increase in histamine-induced [Ca²⁺]_m responses even if the steady-state [Ca²⁺]_er remained unaltered (unpublished data).

Based on previous observations (Gincel et al., 2001; Csordas et al., 2002; Rapizzi et al., 2002; Israelson et al., 2005), we used VDAC1 as the start point for proteomic search of interacting proteins and for unraveling the molecular basis of mitochondrial Ca²⁺ homeostasis. An unexpected, but intriguing, finding of our biochemical studies was the central location of the chaperone grp75 in the interaction between ER and mitochondrial Ca²⁺ channels. grp75, a conserved chaperone, has a well studied role in protein import through the IMM. Still, in yeast mitochondria, mtHsp70/Ssc1 was shown to be significantly more abundant than the translocase (TIM23 complex). Thus, only a small fraction of the protein appears to be involved directly in preprotein translocation (Dekker et al., 1997; Sanjuan Szklarz et al., 2005), suggesting the existence of different pools of the protein. Previous work also reported extramitochondrial localization of grp75 (Ran et al., 2000), and its interaction with extramitochondrial proteins such as the cytosolic p53 or the ER luminal grp94 (Takano et al., 2001; Wadhwa et al., 2002b), although the mechanisms that control the differential sorting of the protein are still completely unknown. According to our immunofluorescence and GFP-tagging studies in HeLa cells grp75 shows complete mitochondrial localization, but obviously cannot be discriminated from an OMM-associated pool. Biochemical studies, however, demonstrate that a matrix-localized pool participates in forming complexes in the 200–400-kD range and represents the major fraction of the total mitochondrial grp75 content, whereas a minor grp75 pool resides in the low-density (MAM) mitochondrial fraction, participating in complexes in the megaDalton range and comprising OMM and ER membrane proteins. To further support an independent function of the nonmatrix pool, we constructed a grp75 mutant lacking the mitochondrial presequence, and thus incompetent for import in the matrix. This protein retained the capacity to enhance mitochondrial Ca²⁺ accumulation, strongly arguing for the notion that this role of grp75 is not only independent from its chaperone activity in the matrix but also depends on a physically separated protein pool.

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.

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 .

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.

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.

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.

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 63×/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.

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.

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.