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

The voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane mediates metabolic flow, Ca 2+ , and cell death signaling between the endoplasmic reticulum (ER) and mitochondrial networks. We demonstrate that VDAC1 is physically linked to the endoplasmic reticulum Ca 2+ -release channel inositol 1,4,5-trisphosphate receptor (IP 3 R) 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 IP 3 R on the ER or mitochondrial surface, which directly enhanced Ca 2+ accumulation in mitochondria. Knockdown of grp75 abolished the stimulatory effect, highlighting chaperone-mediated conformational coupling between the IP 3 R and the mitochondrial Ca 2+ uptake machinery. Because organelle Ca 2+ homeostasis influences fundamentally cellular functions and death signaling, the central location of grp75 may represent an important control point of cell fate and pathogenesis.

VDAC1, grp75, and IP 3 Rs 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 Ca 2+ 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 Ca 2+ -mediated cell death ( Wadhwa et al., 2002a ; Liu et al., 2005 ). Table I. VDAC1 interactors found by yeast two-hybrid screening Name Accession number DnaJ (Hsp40) homologue, subfamily A, member 1; DNAJA1 NM_001539 filamin B, beta (actin-binding protein 278); FLNB NM_001457 heat shock 70-kd protein 5; HSPA5 NM_005347 heat shock 70-kd protein 9b; HSPA9B BC024034 protein phosphatase 1g (formerly 2c), magnesium-dependent, gamma isoform; PPM1G NM_002707 t complex–associated testis-expressed 1-like 1; TCTEL1 D50663 tetratricopeptide repeat domain 1; TCC1 NM_0033114 thioredoxin-like 1; TXNL1 AF052659 tubulin-specific chaperone c; TBCC BC020170 zinc finger–like protein 9; ZPR9 AY046059 Yeast two-hybrid screening was carried using the pLexA system according to the protocol of Gyuris et al. (1993) . For details see Supplementary materials and methods. Approximately 90% of the clones contained a sub-sequence of the ER-resident chaperone heat shock 70-kD protein 5 (HSPA5, grp-78), most probably reflecting the requirement of efficient folding of the VDAC1 protein in yeast. The results of sequencing of the remaining clones are shown in the table. One group of the putative interacting proteins were found to be cytoskeletal and signaling elements (shown in bold); another group (shown in normal) were found to be folding intermediates, presumably underlying the proper function of VDAC1. 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 Ca 2+ 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 Ca 2+ channels from both the OMM (VDAC1) and the ER (IP 3 R; 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 IP 3 R, besides the microsomes, is present in the crude mitochondria and the MAM fractions and is absent in the purified mitochondria ( Fig. 1 A ). Figure 1. IP 3 R, 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 IP 3 Rs 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

Based on our conclusions, we further investigated whether the effect of the N-terminal cytosolic domain of the IP 3 R reflects specific protein–protein interactions at the ER–mitochondrial contacts. We first verified that the effect of IP 3 R-LBD 224-605 on mitochondrial Ca 2+ uptake is independent of IP 3 buffering. For this purpose, we used a point-mutated (K508A) IP 3 R-LBD 224-605 , which is unable to bind IP 3 . The K508 mutant increased the [Ca 2+ ] m rise in a manner similar to the wild-type (although slightly less efficient), but, as expected, did not modify the [Ca 2+ ] c response ( Fig. 2, C and D ). The capacity of an IP 3 -insensitive IP 3 R-LBD 224-605 to enhance mitochondrial Ca 2+ uptake was also confirmed in digitonin-permeabilized HeLa cells. In this case, mitochondrial Ca 2+ uptake is exclusively dictated by the perfused [Ca 2+ ], and it is totally independent of IP 3 R activity. In permeabilized cells, Ca 2+ uptake was triggered by the perfusion of an intracellular buffer containing Ca 2+ 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-IP 3 R-LBD 224-605 increased the rate of mitochondrial Ca 2+ 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 IP 3 binding cannot account for the mitochondrial effect was further confirmed by the demonstration that a structurally unrelated IP 3 -binding protein domain, the PH domain of the PLC-like protein p130 (p130PH; Lin et al., 2005 ), targeted to the OMM, reduced both the [Ca 2+ ] m and [Ca 2+ ] c responses ( Fig. 2 D ). Interestingly, the reduction of the [Ca 2+ ] m response was more pronounced for the OMM-targeted PH domain than for the untargeted cytosolic version of the IP 3 buffer, although the two proteins were equally effective on [Ca 2+ ] c . These data (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200608073/DC1 ) further stress the strict dependence of mitochondrial Ca 2+ homeostasis on the ER– mitochondrial contacts, and thus on the Ca 2+ release occurring in these microdomains. The IP 3 R-LBD 224-605 was also shown to play an important role in the regulation of IP 3 R 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 IP 3 R, targeted to the exterior of the OMM (OMM-IP 3 R 1-604 ), augmented mitochondrial Ca 2+ uptake ( Fig. 2 D ). These results exclude that the stimulatory effect of the IP 3 R-LBD 224-605 was exerted through unmasking this intramolecular interaction in the endogenous IP 3 R; instead, they support a model in which the entire N-terminal IP 3 R exerts direct activation on the mitochondrial Ca 2+ uptake machinery. Finally, we investigated the regulatory activity on mitochondria of the IP 3 R-LBD 224-605 when the [Ca 2+ ] c rise is elicited in the cell by the opening of plasma membrane channels. Under those conditions, not only the [Ca 2+ ] c rise is IP 3 R-independent, but the [Ca 2+ ] c and ensuing [Ca 2+ ] m increases are markedly slower and smaller than upon ER Ca 2+ release. We thus measured [Ca 2+ ] m after emptying the ER Ca 2+ pool with the SERCA blocker tert-butyl-benzohydroquinone (tBHQ) in Ca 2+ -free medium and re-adding CaCl 2 . This protocol induces capacitative Ca 2+ entry, causing a [Ca 2+ ] c rise and subsequent mitochondrial Ca 2+ uptake. As presented in Fig. 4 , IP 3 R-LBD 224-605 –expressing cells showed an ∼60% increase in the influx-dependent [Ca 2+ ] m response (top), even if the [Ca 2+ ] c rise remained unaltered (bottom). This increase in [Ca 2+ ] m was almost doubled, as compared with the effect after histamine-/IP 3 -induced Ca 2+ release from the ER ( Fig. 2 B ). Thus, we concluded that local IP 3 buffering masks the stimulatory effect of the IP 3 R-LBD 224-605 upon ER Ca 2+ release, and, indeed, the effect of the IP 3 R-LBD is established at the ER–mitochondrial contacts. Figure 4. The effect of IP 3 R-LBD 224-605 on mitochondrial Ca 2+ uptake after capacitative Ca 2+ influx. [Ca 2+ ] m and [Ca 2+ ] 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 Ca 2+ -free medium (100 μM KRB-EGTA; 4 min), Ca 2+ influx was induced by the re-addition of 2 mM CaCl 2 to the extracellular medium. (A) Representative traces of control (black traces) and OMM-IP 3 R-LBD 224-605 –cotransfected cells (gray traces) are shown. ([Ca 2+ ] m peak in controls, 12.1 ± 2.11 μM; [Ca 2+ ] m peak in OMM-IP 3 R-LBD 224-605 –expressing cells, 21.2 ± 4.00 μM; P = 0.05; [Ca 2+ ] c peak in controls, 0.96 ± 0.04 μM; [Ca 2+ ] c peak in OMM-IP 3 R-LBD 224-605 –expressing cells, 1.04 ± 0.03 μM). In B, data normalized to the mean ± the SEM of the

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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 [Ca 2+ ] microdomains generated at the mouth of IP 3 Rs. 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 IP 3 R-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 IP 3 R-LBD 224-605 on mitochondrial Ca 2+ uptake. Moreover, the proteomic data also highlight the central role of grp75 in this interaction. VDAC and IP 3 Rs coprecipitate with grp75, and the chaperone is coimmunoprecipitated by both anti-IP 3 R and -VDAC antibodies, indicating that it is the key assembling molecule in the loose interaction between the two ion channels. Within the IP 3 R–grp75–VDAC complex, potentiation of mitochondrial Ca 2+ accumulation by the IP 3 R-LBD 224-605 does not require IP 3 binding, as demonstrated by the fact that it is retained by the K508A mutant, which is unable to bind IP 3 ( Varnai et al., 2005 ). Although the mutant shows the same stimulatory effect ( Fig. 2 ), one should remember that wild-type IP 3 R-LBD 224-605 , because of IP 3 buffering, reduces ER Ca 2+ release, and thus conclude that the wild type is somewhat more effective than the mutant. To further confirm independence from IP 3 buffering, we measured mitochondrial Ca 2+ uptake after capacitative influx through the plasma membrane ( Figs. 4 and 6 ). Also, under those experimental conditions, the IP 3 R-LBD 224-605 potently stimulated mitochondrial Ca 2+ uptake. As for the molecular mechanism of the effect on the mitochondrial Ca 2+ machinery, different scenarios could be envisioned. In the first, the recombinantly expressed IP 3 R-LBD, both from the OMM and ER side, could interact with the endogenous IP 3 R itself, and modify the probability of its interaction with grp75/VDAC. Indeed, it was previously shown that intramolecular interactions between different domains of the IP 3 R, such as the 224–605 minimal IP 3 -binding domain and the 1

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.

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 [Ca 2+ ] values, as previously described ( Chiesa et al., 2001 ). All aequorin measurements were performed in Krebs-Ringer bicarbonate (KRB) containing 1 mM CaCl 2 (KRB/Ca 2+ ; Krebs-Ringer modified buffer: 135 mM NaCl, 5 mM KCl, 1 mM MgSO 4 , 0.4 mM K 2 HPO 4 , 1 mM CaCl 2 , 5.5 mM glucose, and 20 mM Hepes, pH 7.4). [Ca 2+ ] c after capacitative Ca 2+ influx was measured by preincubating HeLa cells with the SERCA blocker tBHQ (100 μM) in a KRB solution containing no Ca 2+ and 100 μM EGTA. Cytoplasmic Ca 2+ signal and mitochondrial Ca 2+ uptake were evoked by adding 2 mM CaCl 2 to the medium. For [Ca 2+ ] er measurements, erAEQmut-transfected cells were reconstituted with coelenterazine n, after ER Ca 2+ depletion in a solution containing 0 [Ca 2+ ], 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.

Table S1 shows [Ca 2+ ] m and [Ca 2+ ] 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 IP 3 R-LBD 224-605 on cytoplasmic Ca 2+ responses and ER Ca 2+ homeostasis. Fig. S3 shows the effect of cytosolic- and OMM-targeted p130-PH domain on mitochondrial Ca 2+ uptake. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200608073/DC1 .