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Mitochondrial Ca 2+  uptake is a vital process that controls distinct cell and organelle functions. Mitochondrial calcium uptake 1 (MICU1) was identified as key regulator of the mitochondrial Ca 2+  uniporter (MCU) that together with the essential MCU regulator (EMRE) forms the mitochondrial Ca 2+  channel. However, mechanisms by which MICU1 controls MCU/EMRE activity to tune mitochondrial Ca 2+  signals remain ambiguous. Here we established a live-cell FRET approach and demonstrate that elevations of cytosolic Ca 2+  rearranges MICU1 multimers with an EC 50  of 4.4 μM, resulting in activation of mitochondrial Ca 2+  uptake. MICU1 rearrangement essentially requires the EF-hand motifs and strictly correlates with the shape of cytosolic Ca 2+  rises. We further show that rearrangements of MICU1 multimers were independent of matrix Ca 2+  concentration, mitochondrial membrane potential, and expression levels of MCU and EMRE. Our experiments provide novel details about how MCU/EMRE is regulated by MICU1 and an original approach to investigate MCU/EMRE activation in intact cells.

Rearrangement of MICU1 multimers for activation of MCU is solely controlled by cytosolic Ca2+