Abstract
Mitochondrial Ca(2+) uptake, a process crucial for bioenergetics and Ca(2+) signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca(2+)-activated Ca(2+) channel, with the Ca(2+) pore formed by the MCU protein and Ca(2+)-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca(2+) 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 Ca(2+) landscape.
Highlights
Ca2+ regulation of key mitochondrial processes such as ATP production and initiation of apoptosis is controlled by precise balance of Ca2+ influx and efflux across the mitochondrial inner membrane (Gunter et al, 2000; Rizzuto et al, 2012)
While analyzing mitochondrial calcium uniporter (MCU)/EMRE from various species (Figure 1C), we noticed that human MCU forms functional Ca2+ channels with human EMRE or with C. elegans EMRE, but C. elegans MCU supports mitochondrial Ca2+ uptake only with ceEMRE (Figure 4D). These results present an opportunity to test which of ceMCU’s two transmembrane helices (TMHs) is responsible for discriminating against hEMRE
A mitochondrial Ca2+ uptake protein 1 (MICU1) mutant carrying an electrostatically neutered sequence (KKKKR =>EQEQR) readily complexes with MICU2, but not with EMRE (Figure 6B). These results strongly argue that the EMRE-MICU1 interaction is mediated by this electrostatic pair
Summary
Ca2+ regulation of key mitochondrial processes such as ATP production and initiation of apoptosis is controlled by precise balance of Ca2+ influx and efflux across the mitochondrial inner membrane (Gunter et al, 2000; Rizzuto et al, 2012). A few years ago, the field witnessed a groundbreaking achievement — identification of the MCU gene (Baughman et al, 2011; De Stefani et al, 2011). The 35-kDa MCU protein oliogomerizes with unknown stoichiometry to form a Ca2+-selective pore (Baughman et al, 2011). MCU possesses two transmembrane helices (TMHs) connected by a short loop that hosts a signature sequence (DIME) thought to contribute to a Ca2+-selective permeation site. The N- and C-terminal regions of MCU are exposed to the mitochondrial matrix, each with a coiled-coil sequence of unknown function
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