Reconstitution of the mitochondrial calcium uniporter in yeast

Abstract

The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits -mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)-and two soluble regulatory components-MICU1 and its paralog MICU2. The minimal components sufficient for in vivo uniporter activity are unknown. Here we consider Dictyostelium discoideum (Dd), a member of the Amoebazoa outgroup of Metazoa and Fungi, and show that it has a highly simplified uniporter machinery. We show that D. discoideum mitochondria exhibit membrane potential-dependent calcium uptake compatible with uniporter activity, and also that expression of DdMCU complements the mitochondrial calcium uptake defect in human cells lacking MCU or EMRE. Moreover, expression of DdMCU in yeast alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Having established yeast as an in vivo reconstitution system, we then reconstituted the human uniporter. We show that coexpression of MCU and EMRE is sufficient for uniporter activity, whereas expression of MCU alone is insufficient. Our work establishes yeast as a powerful in vivo reconstitution system for the uniporter. Using this system, we confirm that MCU is the pore-forming subunit, define the minimal genetic elements sufficient for metazoan and nonmetazoan uniporter activity, and provide valuable insight into the evolution of the uniporter machinery.

Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter

“…The NMR experiments were performed using uniformly [ 15 N, 13 C, 2 H]-labeled cMCU-ΔNTD, which was expressed in Escherichia coli and purified by nickel-nitrilotriacetic acid affinity, ion exchange, and size-exclusion chromatography using a previously established protocol (14) (Fig. 1A).…”

“…Two soluble components in the intermembrane space, MICU1 and MICU2, are Ca 2+ -sensing proteins that gate the activity of MCU based on outside Ca 2+ concentrations through EMRE (9)(10)(11)(12). The current consensus is that the minimum requirements for uniporter-mediated Ca 2+ flux in metazoans are MCU and EMRE, whereas in lower organisms such as Dictyostelium that lack EMRE, MCU is able to operate (13).…”

The mitochondrial Ca2+ uniporter complex

“…MCU was identified by the groups of Mootha and Rizzuto as the ion-conducting pore of the uniporter [41-43, 44]. MCUb, a homolog of MCU, was identified as a dominant-negative regulator of the channel [45].…”

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis

“…The recent identification of several protein families making up the mitochondrial Ca 2+ uniporter complex (MCUC) in mammals, including the pore-forming MCU (MCU and MCUb;Baughman et al, 2011;De Stefani et al, 2011;Raffaello et al, 2013), and the regulatory, associated MICUs (MICU1, 2, and 3; Perocchi et al, 2010;Plovanich et al, 2013), EMRE (Sancak et al, 2013), and MCUR1/CCDC90A (although potentially not directly; Mallilankaraman et al, 2012b;Paupe et al, 2015), has spurred intense research across the biomedical disciplines Mallilankaraman et al, 2012a;Csordás et al, 2013;Hoffman et al, 2013;Marchi et al, 2013;Pan et al, 2013;Raffaello et al, 2013;Kovács-Bogdán et al, 2014;Logan et al, 2014;Patron et al, 2014;Wang et al, 2014). While some genes of the complex components appear not to be present in plants (e.g., MCUb and EMRE), others have multiple homologs (six for the functional poreforming subunit MCU in Arabidopsis [Stael et al, 2012] and maize [Zea mays; Meng et al, 2015] and two for MCUR1/CCDC90A in Arabidopsis), suggesting potential functional modification and/or differentiation.…”