MICU1 encodes a mitochondrial EF hand protein required for Ca(2+) uptake

Abstract

Mitochondrial calcium uptake has a central role in cell physiology by stimulating ATP production, shaping cytosolic calcium transients and regulating cell death. The biophysical properties of mitochondrial calcium uptake have been studied in detail, but the underlying proteins remain elusive. Here we use an integrative strategy to predict human genes involved in mitochondrial calcium entry based on clues from comparative physiology, evolutionary genomics and organelle proteomics. RNA interference against 13 top candidates highlighted one gene, CBARA1, that we call hereafter mitochondrial calcium uptake 1 (MICU1). Silencing MICU1 does not disrupt mitochondrial respiration or membrane potential but abolishes mitochondrial calcium entry in intact and permeabilized cells, and attenuates the metabolic coupling between cytosolic calcium transients and activation of matrix dehydrogenases. MICU1 is associated with the mitochondrial inner membrane and has two canonical EF hands that are essential for its activity, indicating a role in calcium sensing. MICU1 represents the founding member of a set of proteins required for high-capacity mitochondrial calcium uptake. Its discovery may lead to the complete molecular characterization of mitochondrial calcium uptake pathways, and offers genetic strategies for understanding their contribution to normal physiology and disease.

Fig. 1. Targeted RNAi screen for mitochondrial Ca²⁺ uptake

(a) Integrative approach to predict human mitochondrial proteins involved in mitochondrial Ca²⁺ uptake. Numbers represent the subset of human MitoCarta genes with the indicated property. (b) Targeted RNAi screen of mitochondrial Ca²⁺ uptake for 13 of the 18 top candidate genes. Each circle represents one RNAi hairpin expressed in mt-AEQ HeLa cells. Mitochondrial Ca²⁺ uptake, reported as the integral luminescence following 100 µM histamine stimulation, is plotted as a function of cell viability (relative luminescence units, RLU). RNAi hairpins targeting MICU1 are shown in red. (c) Relationship between mitochondrial Ca²⁺ uptake and MICU1 knockdown. The region of the MICU1 cDNA targeted by four distinct hairpins (sh1–sh4) is shown. Integral luminescence normalized to cell number (mean ± s.d., n=8) is plotted as a function of relative MICU1 gene expression using actin as an endogenous control (mean ± s.d., n=3). (d) cDNA rescue of the sh1-MICU1 mitochondrial Ca²⁺ phenotype. Kinetic traces of mitochondrial Ca²⁺ uptake normalized to cell number are shown for control cells (pLKO.1), MICU1-silenced cells, and rescue cells (mean ± s.d., n=3). (e) Quantification of endogenous (3’-UTR) and endogenous + exogenous (CDS) MICU1 expression in control, MICU1 knockdown, and rescue cells measured by qPCR and reported as relative expression using actin as an endogenous control (mean ± s.d., n=3).

Fig. 2. Mitochondrial membrane potential, respiration, and abundance in MICU1-silenced cells

(a) Mitochondrial membrane potential in MICU1-knockdown (sh1-MICU1) and control (sh-GFP) cells measured by JC-1 fluorescence in the presence or absence of the uncoupler CCCP (5 µM). (b) Basal oxygen consumption rate (OCR, pmol O₂/min) in control and knockdown cells. (c) Normalized OCR in MICU1-knockdown and control cells following the addition of the complex V inhibitor oligomycin (Oligo; 0.5 µM), uncoupler CCCP (0.5 µM), and complex III inhibitor antimycin (0.5 µM). Results are mean ± s.d of five independent replicates. (d) mtDNA copy-number relative to nuclear DNA in MICU1-knockdown and control cells. Values are mean+/−s.d of three independent replicates.

Fig. 3. Measurement of mitochondrial Ca²⁺ uptake kinetics in populations of cells, individual cells, and permeabilized cells

(a) Luminescence measurements of mitochondrial Ca²⁺ in MICU1-silenced (sh1-MICU1) and control (pLKO.1) mt-AEQ cells normalized to 30K cells (mean ± s.d., n=3). (b) Luminescence measurements of mitochondrial Ca²⁺ post addition of CaCl₂ in cells pre-treated with thapsigargin (mean ± s.d., n=3). (c) Representative single-cell measurements of mitochondrial Ca²⁺ dynamics in HeLa cells measured by FRET following treatment with histamine. Traces are representative of n=20 control and n=12 knockdown cells. (d) Similar measurements in HeLa cells following stimulation with thapsigargin. Traces are representative of n=15 control and n=11 knockdown cells. (e) Representative traces of mitochondrial Ca²⁺ uptake in digitonin-permeabilized mt-AEQ HeLa cells using Calcium Green-5N (0.5 µM) to measure extramitochondrial Ca²⁺. Arrowheads denote addition of 100 uM pulses of CaCl₂.

Fig. 4. MICU1 is an EF-hand protein localized to mitochondria

(a) Confocal microscopy image of a HeLa cell expressing MICU1-GFP and DsRed2-Mito. (b) Immunoblot analysis of whole cell lysates, crude mitochondria, Percoll purified mitochondria, and mitoplasts isolated from HEK293 cells expressing a COOH-terminus V5-tagged version of MICU1. (c) Domain structure of MICU1 highlighting two evolutionarily conserved EF hands. (d) cDNA rescue of the sh1-MICU1 mitochondrial Ca²⁺ uptake phenotype using a cDNA expressing a wild-type allele of MICU1 or an EF mutant (MICU1mEF) (mean ± s.d., n=3). (e) Quantification of endogenous (3’-UTR) and endogenous + exogenous (CDS) MICU1 expression for the rescue experiment in (d). Relative gene expression is reported as fold change over control cells (pLKO.1) using actin as an endogenous control (mean ± s.d., n=3).

Fig. 5. Contribution of MICU1 to cytosolic Ca²⁺ dynamics and metabolic coupling

(a) Thapsigargin (5 uM) evoked changes in cytosolic [Ca²⁺] measured using a FRET reporter in MICU1-silenced (sh1-MICU1) and control (pLKO.1) cells and reported as a FRET ratio. Traces are representative of n=14 for control and n=16 for knockdown cells. (b) Histamine (200 uM) evoked changes in cytosolic [Ca²⁺] in MICU1-silenced and control cells. Traces are representative of n=28 for control and n=17 for knockdown cells. (c) Single cell measurement of NADH in MICU1-silenced and control cells. Where indicated, cells are challenged with 150 µM histamine (Hist) and 2 µM rotenone (Rot). Traces are representative of n=12 and n=24 knockdown cells.