Systematic mapping of MCU-mediated mitochondrial calcium signaling networks

The Mitochondrial Ca ²⁺ Uniporter Channel (MCUC) allows calcium entry into the mitochondrial matrix to regulate energy metabolism but also cell death. Although, several MCUC components have been identified, the molecular basis of mitochondrial Ca ²⁺ signaling networks and their remodeling upon changes in uniporter activity have not been systematically assessed. Using an unbiased and quantitative proteomic approach, we map the MCUC interactome in HEK293 cells under physiological conditions and upon chronic loss or gain of mitochondrial Ca ²⁺ uptake. Besides all previously known subunits of the uniporter, we identify 89 high-confidence interactors linking MCUC to several mitochondrial complexes and pathways, half of which are currently linked to metabolic, neurological, and immunological diseases. As a proof-of-concept, we validate EFHD1 as a binding partner of MCU, EMRE and MCUB with a MICU1-dependent inhibitory effect on Ca ²⁺ uptake. To investigate compensatory mechanisms and functional consequences of mitochondrial Ca ²⁺ dyshomeostasis, we systematically survey the MCU interactome upon silencing of EMRE, MCUB, MICU1 or MICU2. We observe profound changes in the MCU interconnectivity, whereby downregulation of EMRE reduces the number of MCU interactors of over 10-fold, while silencing of MCUB leads to a wider functional network linking MCU to mitochondrial stress response pathways and cell death. Altogether our study provides a comprehensive map of MCUC protein-protein interactions and a rich, high-confidence resource that can be explored to gain insights into the players and mechanisms involved in calcium signal transduction cascades and their relevance in human diseases.