Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex
- Brandeis University, United States
- Howard Hughes Medical Institute, Brandeis University, United States
Figures
Figure 1
Functional analysis of uniporter in various species.
(A) A representative fluorescence-based Ca2+ flux experiment. (B) Characterization of ME-KO HEK 293 cells. Left: western analysis comparing EMRE, MCU, and actin expression in WT or ME-KO cells. Right…
Figure 2 with 1 supplement
Transmembrane orientation of MCU and EMRE.
(A) Western blot analysis of WT-MCU response to PEGM, in the absence or presence of DDM detergent, with molecular weight marker positions indicated on left. (B) PEGM treatment of WT, I49C, or I97C …
Figure 2—figure supplement 1
Uniporter function supported by indicated EMRE mutants.
https://doi.org/10.7554/eLife.15545.005
Figure 3 with 1 supplement
MCU-interacting residues in EMRE.
(A) EMRE constructs with indicated regions substituted by either the C8 epitope or the WALP helix. MTS: mitochondrial targeting sequence. Green boxes: soluble regions. Pink ovals: polyaspartate …
Figure 3—figure supplement 1
Functional impact of mutations in EMRE’s TM helix.
(A) Expression of indicated EMRE mutants. (B) Ca2+ flux traces in EMRE-KO cells expressing EMRE mutants. (C) Bar chart summarizing Ca2+ uptake activity in cells expressing various EMRE mutants. Dashe…
Figure 4 with 3 supplements
Interactions between EMRE and MCU’s TMH1.
(A) Helical projection diagram summarizing functional impact of Trp substitutions in TMHs of MCU. Trp mutation that reduces Ca2+ uptake by >70% is defined as high impact (red), and <30% as low …
Figure 4—figure supplement 1
Functional impact of mutations in MCU’s TM helices.
(A) Expression of selected MCU mutants. (B–C) The effect of mutations in MCU’s TMH1 (B) or TMH2 (C) on Ca2+ flux via the uniporter. Dashed red line: 30% WT activity.
Figure 4—figure supplement 2
Trp mutations in D. discoideum MCU.
(A) Sequence alignment of TMH1 and TMH2 of human and D. discoideum MCU homologues. Red indicates residues selected for Trp substitution. (B) Characterization of D. discoideum MCU mutants. Left: Ca2+ …
Figure 4—figure supplement 3
Uniporter formation by F77A EMRE and MCU mutants.
(A) Uniporter activity in EMRE-KO cells expressing F77A EMRE and MCU mutants as indicated. Activity is normalized to that produced by WT MCU and WT EMRE. (B) Co-IP test of complex formation by A241W …
Figure 5 with 1 supplement
Localization of MICUs and interaction with the pore region.
(A–B) Carbonate extraction (pH 10.5 or 11.5) of MICU1 at 4°C (A) or MICU2 at room temperature (B) for 1 h, showing membrane pellet (M), proteins extracted into supernatant (S), and control (con) …
Figure 5—figure supplement 1
MICU2 interaction with other subunits in the uniporter complex.
(A) Co-IP experiment showing complex formation of MICU1 and MICU2. MICU2 was used as bait. (B) Co-IP experiments using immobilized MICU2 to pull down MCU or EMRE. (C) Immobilized MICU2 was used to …
Figure 6 with 1 supplement
Functional importance of the MICU1-EMRE interaction.
(A–B) Co-IP experiments with WT- or EQEQ-MICU1 (Flag-tagged) used to pull down WT or mutant EMRE proteins in ME-KO cells. MICU1 was detected using anti-Flag, MICU2 by anti-V5, and ΔN- or ΔC-EMRE by …
Figure 6—figure supplement 1
Biochemical characterization of MICU1 knockdown cells.
(A) the mRNA level of MICU1 in MICU1 knockdown cells compared with WT control, as determined by qPCR. (B) MCU and EMRE expression in these cells.
Figure 7
The physiological role of EMRE.
Cartoon summarizing main findings here, illustrating how the two functions of EMRE, MCU activation and MICU1 retention, together prevent Ca2+ leakage through the uniporter complex in resting …
Additional files
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Supplementary file 1
Supplementary experimental procedure.
- https://doi.org/10.7554/eLife.15545.017
