The Mitochondrial Ca uniporter is a central regulator of interorganellar Ca transfer and NFAT activation.

Mitochondrial Ca uptake tailors the strength of stimulation of plasma membrane phospholipase C-coupled receptors to that of cellular bioenergetics. However, how Ca uptake by the mitochondrial Ca uniporter (MCU) shapes receptor-evoked interorganellar Ca signaling is unknown. Here, we used CRISPR/Cas9 gene knockout, subcellular Ca imaging, and mathematical modeling to show that MCU is a universal regulator of intracellular Ca signaling across mammalian cell types. MCU activity sustains cytosolic Ca signaling by preventing Ca-dependent inactivation of store-operated Ca release-activated Ca channels and by inhibiting Ca extrusion. Paradoxically, MCU knockout (MCU-KO) enhanced cytosolic Ca responses to store depletion. Physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca oscillations, endoplasmic reticulum Ca refilling, nuclear translocation of nuclear factor for activated T cells transcription factors, and cell proliferation, without altering inositol-1,4,5-trisphosphate receptor activity. Our data show that MCU has dual counterbalancing functions at the cytosol-mitochondria interface, whereby the cell-specific MCU-dependent cytosolic Ca clearance and buffering capacity of mitochondria reciprocally regulate interorganellar Ca transfer and nuclear factor for activated T cells nuclear translocation during receptor-evoked signaling. These findings highlight the critical dual function of the MCU not only in the acute Ca buffering by mitochondria but also in shaping endoplasmic reticulum and cytosolic Ca signals that regulate cellular transcription and function.