Mitochondrial Ca²⁺ uptake, a process crucial for bioenergetics and Ca²⁺ signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca²⁺-activated Ca²⁺ channel, with the Ca²⁺ pore formed by the MCU protein and Ca²⁺-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca²⁺ permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca²⁺ landscape.

DOI: http://dx.doi.org/10.7554/eLife.15545.001