title: Shot-gun proteomic analysis of mitochondrial D-loop DNA binding proteins: identification of mitochondrial histones.
abstract: Transcription and replication of mitochondrial DNA (mtDNA) are regulated by nuclear DNA-encoded proteins that are targeted into mitochondria. A decrease in mtDNA copy number results in mitochondrial dysfunction, which may lead to insulin resistance and metabolic syndromes. We analyzed mitochondrial proteins that physically bind to human mitochondrial D-loop DNA using a shot-gun proteomics approach following protein enrichment by D-loop DNA-linked affinity chromatography. A total of 152 D-loop DNA binding proteins were identified by peptide sequencing using ultra high pressure capillary reverse-phase liquid chromatography/tandem mass spectrometry. Bioinformatic analysis showed that 68 were mitochondrial proteins, 96 were DNA/RNA/protein binding proteins and 114 proteins might form a complex via protein-protein interactions. Histone family members of H1, H2A, H2B, H3, and H4, were detected in abundance among them. In particular, histones H2A and H2B were present in the mitochondrial membrane as integral membrane proteins and not bound directly to mtDNA inside the organelle. Histones H1.2, H3 and H4 were associated with the outer mitochondrial membrane. Silencing of H2AX expression inhibited mitochondrial protein transport. Our data suggests that many mitochondrial proteins may reside in multiple subcellular compartments like H2AX and exert multiple functions.
authors.affiliation: Department of Physiology, College of Medicine, Kyung Hee University, Seoul 130-701, South Korea.
authors.forename: Yon-Sik
authors.initials: YS
authors.lastname: Choi
authors.forename: Jae
authors.initials: J
authors.lastname: Hoon Jeong
authors.forename: Hye-Ki
authors.initials: HK
authors.lastname: Min
authors.forename: Hee-Jung
authors.initials: HJ
authors.lastname: Jung
authors.forename: Daehee
authors.initials: D
authors.lastname: Hwang
authors.forename: Sang-Won
authors.initials: SW
authors.lastname: Lee
authors.forename: Youngmi
authors.initials: Y
authors.lastname: Kim Pak
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D056148:Chromatography, Reverse-Phase; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D051336:Mitochondrial Membranes / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D021381:Protein Transport; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D053719:Tandem Mass Spectrometry
chemical_list: D004272:DNA, Mitochondrial; D006657:Histones; D024101:Mitochondrial Proteins; D020543:Proteome; D049452:Green Fluorescent Proteins