MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism

Abstract

Ca(2+) flux across the mitochondrial inner membrane regulates bioenergetics, cytoplasmic Ca(2+) signals and activation of cell death pathways. Mitochondrial Ca(2+) uptake occurs at regions of close apposition with intracellular Ca(2+) release sites, driven by the inner membrane voltage generated by oxidative phosphorylation and mediated by a Ca(2+) selective ion channel (MiCa; ref. ) called the uniporter whose complete molecular identity remains unknown. Mitochondrial calcium uniporter (MCU) was recently identified as the likely ion-conducting pore. In addition, MICU1 was identified as a mitochondrial regulator of uniporter-mediated Ca(2+) uptake in HeLa cells. Here we identified CCDC90A, hereafter referred to as MCUR1 (mitochondrial calcium uniporter regulator 1), an integral membrane protein required for MCU-dependent mitochondrial Ca(2+) uptake. MCUR1 binds to MCU and regulates ruthenium-red-sensitive MCU-dependent Ca(2+) uptake. MCUR1 knockdown does not alter MCU localization, but abrogates Ca(2+) uptake by energized mitochondria in intact and permeabilized cells. Ablation of MCUR1 disrupts oxidative phosphorylation, lowers cellular ATP and activates AMP kinase-dependent pro-survival autophagy. Thus, MCUR1 is a critical component of a mitochondrial uniporter channel complex required for mitochondrial Ca(2+) uptake and maintenance of normal cellular bioenergetics.

Figure 1. RNAi screen identifies MCUR1 as a regulator of mitochondrial Ca²⁺ uptake

Changes in 293T cell cytoplasmic (a) and mitochondrial (b) [Ca²⁺] in response to ionomycin (2.5 μM) were simultaneously measured by fluo-4 and rhod-2 imaging, respectively. Each bar represents one target gene silenced with pooled siRNA. (c) qRT-PCR of MCU, MCUR1 and MICU1 mRNA from mouse tissues (n=3; mean ± s.e.m). (d) qRT-PCR of MCUR1 mRNA from 293T cell clones (n=3; mean ± s.e.m). (e) qRT-PCR of MCUR1 mRNA from HeLa cell clones and of rescued MCUR1 mRNA levels in shHe2 clone (n=3; mean ± s.e.m). The same lentiviral shRNAs were used to generate shHK4 and shHe1 and shHK5 and shHe2, respectively. (f) (Top) MCUR1 protein expression levels and densitometric analysis (n=3; ± s.e.m.). (Bottom) Flag-tagged MCUR1 protein expression in clone shHe2 cells reconstituted with shRNA resistant MCUR1 cDNA plasmid. (g and h) Representative images from movies of HEK 293T NegshRNA or shHK5 cells showing cytosolic (green) and mitochondrial (red) [Ca²⁺] before (left), during (middle) and after (right) ionomycin exposure. Scale bar: 20 μm. (i–p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca²⁺] responses in 293T (i–l) and HeLa (m–p) cells challenged with ionomycin or histamine (100 μM), respectively. (n=3) (i) Wild-type 293T cells. (j) Cells expressing negative shRNA. (k) Clone shHK5 (n=4). (l) Quantification of peak rhod-2 fluorescence. **P < 0.01 (mean ± s.e.m.). (m) HeLa cells expressing negative shRNA. (n) Clone shHe2. (o) Clone shHe2 re-expressing MCUR1 (n=3). (p) Quantification of peak rhod-2 fluorescence. *P < 0.05, **P < 0.01 (mean ± s.e.m.). (q) [Ca²⁺]_c and [Ca²⁺]_m signals evoked by ATP (100 μM) and thapsigargin (Tg, 2 μM) were monitored simultaneously using fura2/AM and mtipcam, respectively in control (upper) and MCUR1 KD (middle) HeLa cells. [Ca²⁺]_c calibrated in nM (black), whereas mtipcam fluorescence is inversely normalized to baseline (F₀/F) (red). (r) Summary mean [Ca²⁺]_c and [Ca²⁺]_m peaks during ATP stimulation (negShRNA n=29; MCUR1 KD n=36 cells,. *P < 0.05 (mean ± s.e.m.). (s) Increase in bath [Ca²⁺] (R_fura2) and [Ca²⁺]_m (R_mipcam) signals in response to CaCl₂ (1 μM) and IP₃ (7.5 μM) addition in permeabilized cells.

Figure 2. MCUR1 is required for Ru360 sensitive mitochondrial Ca²⁺ uptake but independent of mitochondrial Ca²⁺ efflux pathway

Digitonin-permeabilized HeLa cells bathed in intracellular-like solution containing thapsigargin (Tg) were loaded with the ΔΨm indicator JC-1 and the Ca²⁺ indicator Fura2FF, to which pulses of 10 μM Ca²⁺ were added before addition of mitochondrial uncoupler CCCP (carbonyl cyanide m-chloro phenyl hydrazone). Representative traces from three independent experiments depict simultaneous changes of bath [Ca²⁺] and ΔΨm in (a) cells expressing negative shRNA, (b) clone shHe1(c) clone shHe2, (d) clone shHe2 re-expressing MCUR1, and (e) HeLa cells stably over-expressing MCUR1. Under similar conditions, 1 μM Ru360 was added before 10 μM Ca²⁺ pulses until addition of mitochondrial uncoupler CCCP. Representative traces from three independent experiments depict simultaneous changes of bath [Ca²⁺] and ΔΨm in (f) Negative shRNA cells, (g) MCUR1 knockdown clone shHe1 and (h) shHe2, and (i) in control cells over-expressing MCUR1. Neg shRNA (j) and MCUR1 overexpressing (k) HEK 293T cells were permeabilized with digitonin in intracellular-like medium containing thapsigargin (Tg) and bath [Ca²⁺] indicator Fura FF, and then pulsed with 10 μM Ca²⁺. After mitochondrial clearance of bath Ca²⁺, Ru360 caused elevation of bath [Ca²⁺], indicating that steady-state bath [Ca²⁺] after pulse was maintained by balance of MCU-mediated Ca²⁺ uptake and CGP37157 (10 μM)-sensitive Na⁺-Ca²⁺ exchanger-mediated extrusion. CCCP added as indicated. Solid line is mean; shaded areas are ± s.e.m. (n=3). (l) [Ca²⁺]_m efflux rate derived from (j) and (k) during initial 60 s following Ru360 addition (n.s.; not significant, (n=3)). (m) HEK 293T cells stably expressing negative shRNA or MCUR1 shRNA (clone shHe2) and clone ShHe2 re-expressing MCUR1 cells were permeabilized with digitonin in intracellular-like medium containing bath [Ca²⁺] indicator Fura FF. CCCP added as indicated. Traces show bath [Ca²⁺] (μM). (solid lines are mean; shaded regions are ± s.e.m. (n= 3). (n) Quantification of total mitochondrial Ca²⁺ released after CCCP addition. *P < 0.05, **P < 0.01 (mean ± s.e.m).

Figure 3. Mitochondrial inner membrane localization and topology of MCUR1 and its interaction with MCU

Confocal images of HeLa cells transiently co-transfected for 48 hrs with (a) GFP-tagged MCUR1 and DsRed Mito plasmids or (b) mRFP-tagged MCUR1 and EYFP-Mito. Scale bar: 20 μm. (c) Immunoblot analysis of Flag-tagged MCUR1 in HeLa cell crude mitochondrial fraction, outer mitochondrial membrane (OMM) and mitoplasts, using antibodies against VDAC1 (OMM protein), COII (integral membrane marker) and Flag. (d) Immunoblot analyses of mitochondria-containing pellet and cytosolic fractions from plasma membrane-permeabilized HeLa cells. Permeabilized cells were treated with or without tBid (50 nM) for outer mitochondrial membrane (OMM) permeabilization and appearance of cytosolic cytochrome c was verified. Intact and OMM permeabilized samples were exposed to Proteinase K for 10 min. These samples were probed using antibodies against HSP60, OXA1, Flag and MCUR1. (e) Reciprocal co-immunoprecipitation of MCU-GFP and MCUR1-Flag transiently expressed in COS7 cells. Representative of four independent experiments. (f) Co-immunoprecipitation of MICU1-Flag with MCU-GFP but not with MCUR1-GFP transiently expressed in COS7 cells. Representative of four independent experiments. (g) Immunoprecipitation of LETM1-GFP with anti-LETM1 failed to pull down MCUR1-Flag in transiently-transfected COS7 cells. (h) Immunoprecipitation with Flag antibody pulled down LETM1-Flag or MCU-Flag transiently expressed in COS7 cells (IP lanes 2 and 3, lower panel), but only co-immunoprecipitated MCUR1-V5 in the MCU-Flag expressing cells (IP lanes 4 vs 5, upper panel), despite lower expression of MCU-Flag in MCUR1-cotransfected cells (compare MCU-Flag and LETM1-Flag expression in MCUR1-cotransfected cells in lysate lanes 4 and 5, bottom panel). Representative of three independent experiments.

Figure 4. MCUR1 is essential for MCU-dependent mitochondrial Ca²⁺ uptake

(a) qRT-PCR of MCU mRNA from wild type and stable MCUR1 over-expressing HeLa cells that were transiently transfected with scrambled siRNA or siRNA against MCU. ***P < 0.001 (mean ± s.e.m). (b) [Ca²⁺]_m responses to histamine (100 μM) in HeLa cells stably over-expressing MCUR1 and in cells transiently transfected with scrambled siRNA or MCU siRNA, and in stable MCUR1 over-expressing HeLa cells transfected with MCU siRNA. After 48 hr of siRNA transfection, cells were loaded with rhod-2 and [Ca²⁺]_m responses were visualized by confocal microscopy. (solid lines are mean; shaded regions are ± s.e.m.; n= 3). (c) Quantification of peak rhod-2 fluorescence following histamine stimulation. *P < 0.05, ***P < 0.001 (mean ± s.e.m; n=3). (d) qRT-PCR of MCU mRNA from wild type and MCUR1 knockdown HeLa cells that were transiently transfected with MCU cDNA. **P < 0.01, ***P < 0.001 (mean ± s.e.m.; n=3). (e) [Ca²⁺]_m responses to histamine (100μM) in wild-type and MCUR1 (shHe2) knockdown HeLa cells over-expressing MCU. Negative shRNA and MCUR1-shHe2 cells were used as controls. (solid lines are mean; shaded regions are ± s.e.m.; n= 3). (f) Quantification of peak rhod-2 fluorescence following histamine stimulation. ***P < 0.001 (mean ± s.e.m; n=3).

Figure 5. MCUR1 is required for maintenance of cellular bioenergetics

(a) AMP/ATP ratios in stable HeLa cell lines stably expressing negative shRNA, MCUR1 shRNA (clone shHe2) or ShHe2 with MCUR1 re-expressed. **P < 0.01 (mean ± s.e.m.; n=3). (b) O₂ consumption rates (OCR) in stable HeLa cells expressing irrelevant shRNA, clone ShHe2, and clone ShHe2 re-expressing MCUR1, exposed sequentially to (a) oligomycin, (b) FCCP, and (c) rotenone plus myzothiazol. (c) Basal and maximal OCR in cells as described in (B). *P < 0.05 (mean ± s.e.m.; n=3). (d) Western blot of phosphorylated and total AMPK (top) and densitometric analysis (bottom) in stable HeLa lines expressing negative shRNA or MCUR1 shRNA (clone sheHe2) and clone shHe2 re-expressing MCUR1. *P < 0.05, **P < 0.01 (mean ± s.e.m.; n=3). (e) Western blot of LC3 or tubulin in stable HeLa lines expressing negative shRNA or MCUR1 shRNA (clone sheHe2) and clone ShHe2 re-expressing MCUR1 (top) and quantification of LC3-II/(LC3-I + LC3-II) (bottom) expressed as fold increase over levels in cells expressing irrelevant shRNA. *P < 0.05, **P < 0.01 (mean ± s.e.m.; n=3). (f and g), as in (d and e). Activation of AMPK (f) and autophagy (**P < 0.001; mean ± s.e.m.; n=3) (g) in absence and presence of InsP₃R inhibitor Xestospongin B (XeB). *P < 0.05, **P < 0.001, ns = not significant (mean ± s.e.m.; n=3).