title: Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure.
abstract: The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.
authors.affiliation: Nijmegen Centre for Mitochondrial Disorders, RadboudUMC, Nijmegen, The Netherlands.|Institute of Molecular and Cell Biology, University of Tartu, Estonia.
authors.forename: Joachim M
authors.initials: JM
authors.lastname: Gerhold
authors.affiliation: Nijmegen Centre for Mitochondrial Disorders, RadboudUMC, Nijmegen, The Netherlands.
authors.forename: Şirin
authors.initials: Ş
authors.lastname: Cansiz-Arda
authors.affiliation: Faculty of Science and Engineering, Biochemistry. Åbo Akademi, Turku, Finland.|Institute of Molecular and Cell Biology, University of Tartu, Estonia.
authors.forename: Madis
authors.initials: M
authors.lastname: Lõhmus
authors.affiliation: Faculty of Science and Engineering, Biochemistry. Åbo Akademi, Turku, Finland.
authors.forename: Oskar
authors.initials: O
authors.lastname: Engberg
authors.affiliation: MRC Mitochondrial Biology Unit, Cambridge, UK.
authors.forename: Aurelio
authors.initials: A
authors.lastname: Reyes
authors.affiliation: Nijmegen Centre for Mitochondrial Disorders, RadboudUMC, Nijmegen, The Netherlands.
authors.forename: Helga
authors.initials: H
authors.lastname: van Rennes
authors.affiliation: Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, University of Newcastle, Newcastle upon Tyne, NE4 5PL, UK.
authors.forename: Alberto
authors.initials: A
authors.lastname: Sanz
authors.affiliation: Institute for Cell and Molecular Biosciences, Newcastle University Institute for Ageing, University of Newcastle, Newcastle upon Tyne, NE4 5PL, UK.
authors.forename: Ian J
authors.initials: IJ
authors.lastname: Holt
authors.affiliation: Faculty of Science and Engineering, Biochemistry. Åbo Akademi, Turku, Finland.
authors.forename: Helen M
authors.initials: HM
authors.lastname: Cooper
authors.affiliation: Nijmegen Centre for Mitochondrial Disorders, RadboudUMC, Nijmegen, The Netherlands.
authors.forename: Johannes N
authors.initials: JN
authors.lastname: Spelbrink
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics; D001692:Biological Transport; D002460:Cell Line; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism*; D004272:DNA, Mitochondrial / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism; D011485:Protein Binding
chemical_list: D004272:DNA, Mitochondrial; C088548:IMMT protein, human; D024101:Mitochondrial Proteins; D009124:Muscle Proteins; D002784:Cholesterol; D000251:Adenosine Triphosphatases