title: Analysis of the structure and function of EMRE in a yeast expression system.
abstract: The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel, and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression system for the mammalian calcium uniporter subunits is useful for investigating their functions. We here established a yeast expression system for the native-form mouse MCU and 4 other subunits. This expression system enabled us to precisely reconstitute the properties of the mammalian MCU complex in yeast mitochondria. Using this expression system, we analyzed the essential MCU regulator (EMRE), which is a key subunit for Ca(2+) uptake but whose functions and structure remain unclear. The topology of EMRE was revealed: its N- and C-termini projected into the matrix and the inter membrane space, respectively. The expression of EMRE alone was insufficient for Ca(2+) uptake; and co-expression of MCU with EMRE was necessary. EMRE was independent of the protein levels of other subunits, indicating that EMRE was not a protein-stabilizing factor. Deletion of acidic amino acids conserved in EMRE did not significantly affect Ca(2+) uptake; thus, EMRE did not have basic properties of ion channels such as ion-selectivity filtration and ion concentration. Meanwhile, EMRE closely interacted with the MCU on both sides of the inner membrane, and this interaction was essential for Ca(2+) uptake. This close interaction suggested that EMRE might be a structural factor for opening of the MCU-forming pore.
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan. Electronic address: tyamamo@genome.tokushima-u.ac.jp.
authors.forename: Takenori
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Yamagoshi
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Harada
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
authors.forename: Mayu
authors.initials: M
authors.lastname: Kawano
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Minami
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Ido
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
authors.forename: Kana
authors.initials: K
authors.lastname: Kuwahara
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Fujita
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
authors.forename: Mizune
authors.initials: M
authors.lastname: Ozono
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Watanabe
authors.affiliation: School of Dentistry, University of Tokushima, Kuramotocho-3, Tokushima 770-8504, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Yamada
authors.affiliation: Niigata University of Pharmacy and Applied Life Sciences, Niigata City 956-8603, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Terada
authors.affiliation: Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan; Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan. Electronic address: yshinoha@genome.tokushima-u.ac.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Shinohara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
chemical_list: D015220:Calcium Channels; C000601188:MCUR1 protein, human; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; C034703:mitochondrial calcium uniporter; D002118:Calcium
keywords: Calcium uniporter; EMRE; MCU; Mitochondria; Yeast