scholarly journals Molecular basis of EMRE-dependence of the human mitochondrial calcium uniporter

2019 ◽  
Author(s):  
Melissa J.S. MacEwen ◽  
Andrew L. Markhard ◽  
Mert Bozbeyoglu ◽  
Forrest Bradford ◽  
Olga Goldberger ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Molecular Basis ◽  
Transmembrane Protein ◽  
Transmembrane Domains ◽  
Chimeric Proteins ◽  
Evolutionary Divergence ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Channel Opening ◽  
Strict Requirement

ABSTRACTThe mitochondrial uniporter is calcium-activated calcium channel complex critical for cellular signaling and bioenergetics. MCU, the pore-forming subunit of the uniporter, contains two transmembrane domains and is found in all major eukaryotic taxa. In amoeba and fungi, MCU homologs are sufficient to form a functional calcium channel, whereas human MCU exhibits a strict requirement for the metazoan-specific, single-pass transmembrane protein EMRE for conductance. Here, we exploit this evolutionary divergence to decipher the molecular basis of the human MCU’s dependence on EMRE. By systematically generating chimeric proteins that consist of EMRE-independent D. discoideum MCU (DdMCU) and H. sapiens MCU (HsMCU), we converged on a stretch of 10 amino acids in DdMCU that can be transplanted to HsMCU to render it EMRE-dependent. We call this region in human MCU the EMRE-dependence domain (EDD). Crosslinking experiments show that HsEMRE directly interacts with MCU at both of its transmembrane domains as well as the EDD. Based on previously published structures of fungal MCU homologs, the EDD segment is located distal to the calcium pore’s selectivity filter and appears flexible. We propose that EMRE stabilizes EDD of MCU, permitting both channel opening and calcium conductance

scholarly journals Evolutionary divergence reveals the molecular basis of EMRE dependence of the human MCU

2020 ◽  
Vol 3 (10) ◽  
pp. e202000718 ◽  
Author(s):  
Melissa JS MacEwen ◽  
Andrew L Markhard ◽  
Mert Bozbeyoglu ◽  
Forrest Bradford ◽  
Olga Goldberger ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Molecular Basis ◽  
Homo Sapiens ◽  
Transmembrane Domains ◽  
Chimeric Proteins ◽  
Evolutionary Divergence ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Channel Opening ◽  
Strict Requirement

The mitochondrial calcium uniporter (MCU) is a calcium-activated calcium channel critical for signaling and bioenergetics. MCU, the pore-forming subunit of the uniporter, contains two transmembrane domains and is found in all major eukaryotic taxa. In amoeba and fungi, MCU homologs are sufficient to form a functional calcium channel, whereas human MCU exhibits a strict requirement for the metazoan protein essential MCU regulator (EMRE) for conductance. Here, we exploit this evolutionary divergence to decipher the molecular basis of human MCU’s dependence on EMRE. By systematically generating chimeric proteins that consist of EMRE-independent Dictyostelium discoideum MCU and Homo sapiens MCU (HsMCU), we converged on a stretch of 10 amino acids in D. discoideum MCU that can be transplanted to HsMCU to render it EMRE independent. We call this region in human MCU the EMRE dependence domain (EDD). Crosslinking experiments show that EMRE directly interacts with HsMCU at its transmembrane domains as well as the EDD. Our results suggest that EMRE stabilizes the EDD of MCU, permitting both channel opening and calcium conductance, consistent with recently published structures of MCU-EMRE.

scholarly journals Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1

2013 ◽  
Vol 143 (1) ◽  
pp. 67-73 ◽  
Author(s):  
Ming-Feng Tsai ◽  
Dawei Jiang ◽  
Linlin Zhao ◽  
David Clapham ◽  
Christopher Miller
Keyword(s):  
Transport Mechanism ◽  
Turnover Rate ◽  
Leucine Zipper ◽  
Transmembrane Protein ◽  
Ruthenium Red ◽  
Mitochondrial Calcium Uniporter ◽  
Mitochondrial Inner Membrane ◽  
Calcium Uniporter ◽  
Ef Hand ◽  
Inner Membrane Protein

The leucine zipper, EF hand–containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca2+ and K+ homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca2+ fluorophore and 45Ca2+-based assays, we demonstrate directly that Letm1 is a Ca2+ transporter, with apparent affinities of cations in the sequence of Ca2+ ≈ Mn2+ > Gd3+ ≈ La3+ > Sr2+ >> Ba2+, Mg2+, K+, Na+. Kinetic analysis yields a Letm1 turnover rate of 2 Ca2+/s and a Km of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca2+/2 H+ antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H+-dependent Ca2+ transport mechanism identified in intact mitochondria.

scholarly journals Structural characterization of the N-terminal domain of the Dictyostelium discoideum mitochondrial calcium uniporter

2019 ◽  
Author(s):  
Yuan Yuan ◽  
Chan Cao ◽  
Maorong Wen ◽  
Min Li ◽  
Ying Dong ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dictyostelium Discoideum ◽  
Self Assembly ◽  
Calcium Influx ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Terminal Domain ◽  
Calcium Uniporter

AbstractThe mitochondrial calcium uniporter (MCU) plays a critical role in the mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multi-component system including the pore-forming subunit MCU and several regulators (MICU1, MICU2, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE. Dictyostelium discoideum (Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologs. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as oligomers, most probably as a pentamer or hexamer. Mutagenesis showed that the acidic residues Asp60, Glu72 and Glu74, which appeared to mediate the parallel interface as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex readily dissociated to lower-order oligomers in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

Abstract 513: Emre Regulates the Mitochondrial Calcium Uniporter in vivo

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Julia C Liu ◽  
Nicole Syder ◽  
Nima Ghorashi ◽  
Thomas B Willingham ◽  
Randi J Parks ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Transmembrane Protein ◽  
Ischemia Reperfusion ◽  
Strenuous Exercise ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Isolated Mitochondria ◽  
Mendelian Genetics ◽  
Calcium Uniporter

Mitochondrial uptake of Ca 2+ plays critical roles in cardiac energy production as well as cell death. The mitochondrial calcium uniporter in mice and humans is a multi-protein complex that includes the channel-forming protein MCU and several other subunit proteins, including EMRE. EMRE is a single transmembrane protein that is conserved among metazoan species and is known to be essential for mitochondrial Ca 2+ uptake in cell culture. To investigate EMRE’s role in organismal physiology, we generated a mouse model of global germline EMRE deletion. We show that EMRE is indeed required for mitochondrial calcium uniporter function in isolated mitochondria from multiple tissues. Although the birth rate of Emre -/- mice is lower than expected by Mendelian genetics (~5-10% instead of ~25%), the mice that are born are viable and appear healthy. Oxygen consumption in isolated mitochondria and cells is not significantly affected by loss of EMRE, and similarly the mice do not exhibit overt metabolic impairment, even under strenuous exercise. No significant differences between Emre -/- and wild-type ( WT ) cardiac function at baseline and after isoproterenol stimulation are evident by echocardiography. Moreover, Emre -/ - hearts are not protected from ischemia/reperfusion injury in a Langendorff perfusion model (mean infarct area 61% in Emre -/- hearts; 57% in WT ). Collectively, these data and their similarities to results found via germline Mcu deletion demonstrate that EMRE is indeed essential for mitochondrial Ca 2+ uptake in vivo. Furthermore, we find evidence that EMRE protein expression is elevated in some mouse muscular dystrophy models, suggesting that modulation of EMRE levels may play a role in regulating uniporter activity in conditions of stress or disease. We therefore further explore whether and how EMRE expression changes with isoproterenol-induced cardiac hypertrophy in mice and in samples from human patients with heart failure. Understanding of how uniporter components such as EMRE can regulate MCU in a diseased state can inform better therapeutic strategies aimed at restoring mitochondrial metabolic homeostasis.

288-LB: Effects of the Hepatic Mitochondrial Calcium Uniporter on Hepatic Gluconeogenesis and Mitochondrial Metabolism in Awake Mice

Diabetes ◽  
2019 ◽  
Vol 68 (Supplement 1) ◽  
pp. 288-LB
Author(s):  
JI EUN LEE ◽  
LEIGH GOEDEKE ◽  
YE ZHANG ◽  
RACHEL J. PERRY ◽  
RUSSELL GOODMAN ◽  
...  
Keyword(s):  
Mitochondrial Metabolism ◽  
Mitochondrial Calcium ◽  
Hepatic Gluconeogenesis ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter

Cobalt amine complexes and Ru265 interact with the DIME region of the mitochondrial calcium uniporter

2021 ◽  
Author(s):  
Joshua J. Woods ◽  
Madison X. Rodriguez ◽  
Chen-Wei Tsai ◽  
Ming-Feng Tsai ◽  
Justin J. Wilson
Keyword(s):  
Mechanisms Of Action ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Amine Complexes ◽  
Calcium Uniporter

The MCU-inhibitory properties and mechanisms of action of Co3+ amine complexes and Ru265 are described.

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