title: 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.
authors.affiliation: Howard Hughes Medical Institute andDepartment of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114;Department of Systems Biology, Harvard Medical School, Boston, MA 02446;
authors.forename: Erika
authors.initials: E
authors.lastname: Kovács-Bogdán
authors.affiliation: Howard Hughes Medical Institute andDepartment of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114;Department of Systems Biology, Harvard Medical School, Boston, MA 02446;
authors.forename: Yasemin
authors.initials: Y
authors.lastname: Sancak
authors.affiliation: Howard Hughes Medical Institute andDepartment of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114;Department of Systems Biology, Harvard Medical School, Boston, MA 02446;
authors.forename: Kimberli J
authors.initials: KJ
authors.lastname: Kamer
authors.affiliation: Howard Hughes Medical Institute andDepartment of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114;Department of Systems Biology, Harvard Medical School, Boston, MA 02446;
authors.forename: Molly
authors.initials: M
authors.lastname: Plovanich
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114;Department of Genetics, Harvard Medical School, Boston, MA 02446;
authors.forename: Ashwini
authors.initials: A
authors.lastname: Jambhekar
authors.affiliation: Department of Neurology, Massachusetts General Hospital, Boston, MA 02114;Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114; and.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Huber
authors.affiliation: Department of Neurology, Massachusetts General Hospital, Boston, MA 02114;Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114; and.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Myre
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114;Department of Genetics, Harvard Medical School, Boston, MA 02446;
authors.forename: Michael D
authors.initials: MD
authors.lastname: Blower
authors.affiliation: Howard Hughes Medical Institute andDepartment of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114;Department of Systems Biology, Harvard Medical School, Boston, MA 02446;Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114; andBroad Institute, Cambridge, MA 02142 vamsi@hms.harvard.edu.
authors.forename: Vamsi K
authors.initials: VK
authors.lastname: Mootha
mesh_terms: D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry*; D002460:Cell Line; D004023:Dictyostelium; D005821:Genetic Techniques; D057809:HEK293 Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D033681:Mitochondrial Membrane Transport Proteins; C034703:mitochondrial calcium uniporter; D002118:Calcium