Variable Assembly of EMRE and MCU Creates Functional Channels with Distinct Gatekeeping Profiles.
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ABSTRACT:
SUBMITTER: Payne R
PROVIDER: S-EPMC7170992 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The mitochondrial calcium uniporter is a multi-subunit Ca2+-activated Ca2+ channel, made up of the pore-forming MCU protein, a metazoan-specific EMRE subunit, and MICU1/MICU2, which mediate Ca2+ activation. It has been established that metazoan MCU requires EMRE binding to conduct Ca2+, but how EMRE promotes MCU opening remains unclear. Here, we demonstrate that EMRE controls MCU activity via its transmembrane helix, while using an N-terminal PKP motif to strengthen binding with MCU. Opening of MCU requires hydrophobic interactions mediated by MCU residues near the pore's luminal end. Enhancing these interactions by single mutation allows human MCU to transport Ca2+ without EMRE. We further show that EMRE may facilitate MCU opening by stabilizing the open state in a conserved MCU gating mechanism, present also in non-metazoan MCU homologs. These results provide insights into the evolution of the uniporter machinery and elucidate the mechanism underlying the physiologically crucial EMRE-dependent MCU activation process.
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Project description: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.
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Project description:The proteins MCU and EMRE form the minimal functional unit of the mitochondrial calcium uniporter complex in metazoans, a highly selective and tightly controlled Ca2+ channel of the inner mitochondrial membrane that regulates cellular metabolism. Here we present functional reconstitution of an MCU-EMRE complex from the red flour beetle, Tribolium castaneum, and a cryo-EM structure of the complex at 3.5 Å resolution. Using a novel assay, we demonstrate robust Ca2+ uptake into proteoliposomes containing the purified complex. Uptake is dependent on EMRE and also on the mitochondrial lipid cardiolipin. The structure reveals a tetrameric channel with a single ion pore. EMRE is located at the periphery of the transmembrane domain and associates primarily with the first transmembrane helix of MCU. Coiled-coil and juxtamembrane domains within the matrix portion of the complex adopt markedly different conformations than in a structure of a human MCU-EMRE complex, suggesting that the structures represent different conformations of these functionally similar metazoan channels.
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Project description:The mitochondrial uniporter (MCU) is an ion channel that mediates Ca(2+) uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca(2+) signaling. Matrix Ca(2+) concentration is similar to that in cytoplasm, despite an enormous driving force for entry, but the mechanisms that prevent mitochondrial Ca(2+) overload are unclear. Here, we show that MCU channel activity is governed by matrix Ca(2+) concentration through EMRE. Deletion or charge neutralization of its matrix-localized acidic C terminus abolishes matrix Ca(2+) inhibition of MCU Ca(2+) currents, resulting in MCU channel activation, enhanced mitochondrial Ca(2+) uptake, and constitutively elevated matrix Ca(2+) concentration. EMRE-dependent regulation of MCU channel activity requires intermembrane space-localized MICU1, MICU2, and cytoplasmic Ca(2+). Thus, mitochondria are protected from Ca(2+) depletion and Ca(2+) overload by a unique molecular complex that involves Ca(2+) sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.
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Project description:The ubiquitous Ca2+ release-activated Ca2+ (CRAC) channel is crucial to many physiological functions. Both gain and loss of CRAC function is linked to disease. While ORAI1 is a crucial subunit of CRAC channels, recent evidence suggests that ORAI2 and ORAI3 heteromerize with ORAI1 to form native CRAC channels. Furthermore, ORAI2 and ORAI3 can form CRAC channels independently of ORAI1, suggesting diverse native CRAC stoichiometries. Yet, most available CRAC modifiers are presumed to target ORAI1 with little knowledge of their effects on ORAI2/3 or heteromers of ORAIs. Here, we used ORAI1/2/3 triple-null cells to express individual ORAI1, ORAI2, ORAI3 or ORAI1/2/3 concatemers. We reveal that GSK-7975A and BTP2 essentially abrogate ORAI1 and ORAI2 activity while causing only a partial inhibition of ORAI3. Interestingly, Synta66 abrogated ORAI1 channel function, while potentiating ORAI2 with no effect on ORAI3. CRAC channel activities mediated by concatenated ORAI1-1, ORAI1-2 and ORAI1-3 dimers were inhibited by Synta66, while ORAI2-3 dimers were unaffected. The CRAC enhancer IA65 significantly potentiated ORAI1 and ORAI1-1 activity with marginal effects on other ORAIs. Further, we characterized the profiles of individual ORAI isoforms in the presence of Gd3+ (5μM), 2-APB (5 μM and 50 μM), as well as changes in intracellular and extracellular pH. Our data reveal unique pharmacological features of ORAI isoforms expressed in an ORAI-null background and provide new insights into ORAI isoform selectivity of widely used CRAC pharmacological compounds.
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Project description:Ca2+ entry into mitochondria is mediated by the Ca2+ uniporter-channel complex containing MCU, the Ca2+-selective pore, and associated regulatory proteins. The roles of MICU proteins are controversial. MICU1 was proposed to be necessary for MCU activity, whereas subsequent studies suggested it inhibits the channel in the low-cytoplasmic Ca2+ ([Ca2+]c) regime, a mechanism referred to as "gatekeeping," that imposes a [Ca2+]c threshold for channel activation at ?1-3 ?M. Here, we measured MCU activity over a wide range of quantitatively controlled and recorded [Ca2+]c. MICU1 alone can mediate gatekeeping as well as highly cooperative activation of MCU activity, whereas the fundamental role of MICU2 is to regulate the threshold and gain of MICU1-mediated inhibition and activation of MCU. Our results provide a unifying model for the roles of the MICU1/2 heterodimer in MCU-channel regulation and suggest an evolutionary role for MICU2 in spatially restricting Ca2+ crosstalk between single inositol 1,4,5-trisphosphate receptor (InsP3R) and MCU channels.
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Project description:The antibiotic andrimid, a nanomolar inhibitor of bacterial acetyl coenzyme A carboxylase, is generated on an unusual polyketide/nonribosomal peptide enzyme assembly line in that all thiolation (T) domains/small-molecule building stations are on separate proteins. In addition, a transglutaminase homologue is used to condense andrimid building blocks together on the andrimid assembly line. The first two modules of the andrimid assembly line yields an octatrienoyl-beta-Phe-thioester tethered to the AdmI T domain, with amide bond formation carried out by a free-standing transglutaminase homologue AdmF. Analysis of the aminomutase AdmH reveals its specific conversion from l-Phe to (S)-beta-Phe, which in turn is activated by AdmJ and ATP to form (S)-beta-Phe-aminoacyl-AMP. AdmJ then transfers the (S)-beta-Phe moiety to one of the free-standing T domains, AdmI, but not AdmA, which instead gets loaded with an octatrienoyl group by other enzymes. AdmF, the amide synthase, will accept a variety of acyl groups in place of the octatrienoyl donor if presented on either AdmA or AdmI. AdmF will also use either stereoisomer of phenylalanine or beta-Phe when presented on AdmA and AdmI, but not when placed on noncognate T domains. Further, we show the polyketide synthase proteins responsible for the polyunsaturated acyl cap can be bypassed in vitro with N-acetylcysteamine as a low-molecular-weight acyl donor to AdmF and also in vivo in an Escherichia coli strain bearing the andrimid biosynthetic gene cluster with a knockout in admA.
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Project description:Basaltic crystal cargoes often preserve records of mantle-derived chemical variability that have been erased from their carrier liquids by magma mixing. However, the consequences of mixing between similarly primitive but otherwise chemically variable magmas remain poorly understood despite ubiquitous evidence of chemical variability in primary melt compositions and mixing-induced disequilibrium within erupted crystal cargoes. Here we report observations from magma-magma reaction experiments performed on analogues of primitive Icelandic lavas derived from distinct mantle sources to determine how their crystal cargoes respond to mixing-induced chemical disequilibrium. Chemical variability in our experimental products is controlled dominantly by major element diffusion in the melt that alters phase equilibria and triggers plagioclase resorption within regions that were initially plagioclase saturated. Isothermal mixing between chemically variable basaltic magmas may therefore play important but previously underappreciated roles in creating and modifying crystal cargoes by unlocking plagioclase-rich mushes and driving resorption, (re-)crystallisation and solid-state diffusion.
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Project description:ObjectiveTo identify distinct behavioral phenotypes of behavioral-variant frontotemporal dementia (bvFTD) and to elucidate differences in functional, neuroimaging, and progression to residential care placement.MethodsEighty-eight patients with bvFTD were included in a cluster analysis applying levels of disinhibition and apathy (Cambridge Behavioural Inventory-Revised) to identify phenotypic subgroups. Between-group (Kruskal-Wallis, Mann-Whitney U) functional differences (Disability Assessment for Dementia) and time to residential care placement (survival analyses) were examined. Cortical thickness differences (whole-brain MRI) were analyzed in patients with bvFTD vs healthy controls (n = 30) and between phenotypic subgroups.ResultsFour phenotypic subgroups were identified: primary severe apathy (n = 26), severe apathy and disinhibition (n = 26), mild apathy and disinhibition (n = 27), and primary severe disinhibition (n = 9). Patients with severely apathetic phenotypes were more functionally impaired and had more extensive brain atrophy than those with mild apathy or severe disinhibition alone. Further imaging analyses indicated that the right middle temporal region is critical for the development of disinhibition, an association that remains with disease progression and in the context of severe apathy. Finally, no difference in time to residential care admission was found between phenotypes.ConclusionsThis study reveals that different clinical behavioral phenotypes of bvFTD have differing profiles of functional decline and distinct patterns of associated cortical changes. These findings emphasize the importance of apathy in functional impairment, highlight the role of the right temporal region in disinhibition, and suggest that disability may be a sensitive outcome measure for treatments targeting reduction of apathy. These phenotypes could also support understanding of prognosis and clinical management.
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