ABSTRACT
Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.
Subject(s)
Mitochondria/genetics , Mitochondrial Proteins/biosynthesis , Nucleoproteins/physiology , Protein Biosynthesis , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/physiology , Cell Line, Tumor , DNA, Mitochondrial/metabolism , HEK293 Cells , Humans , Membrane Proteins/metabolism , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/physiology , Nuclear Proteins/physiology , Prohibitins , RNA/analysis , RNA/isolation & purification , RNA, Messenger/analysis , RNA, Mitochondrial , Repressor Proteins/physiology , Ribosomes/metabolismABSTRACT
Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and ß-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of ß-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some ß-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.
Subject(s)
Actins/physiology , DNA, Mitochondrial/metabolism , Myosin Heavy Chains/physiology , Nonmuscle Myosin Type IIA/physiology , Nonmuscle Myosin Type IIB/physiology , Actins/analysis , Actins/antagonists & inhibitors , Animals , Cells, Cultured , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/isolation & purification , Gene Silencing , Humans , Mice , Mitochondria/chemistry , Mitochondria/ultrastructure , Mitochondrial Proteins/isolation & purification , Myosin Heavy Chains/antagonists & inhibitors , Nonmuscle Myosin Type IIA/analysis , Nonmuscle Myosin Type IIA/antagonists & inhibitors , Nonmuscle Myosin Type IIB/antagonists & inhibitors , RatsABSTRACT
The accessory subunit of mitochondrial DNA polymerase gamma, POLGbeta, functions as a processivity factor in vitro. Here we show POLGbeta has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGbeta increased nucleoid numbers, whereas over-expression of POLGbeta reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGbeta altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGbeta preferentially bound to plasmids with a short displacement-loop, in contrast to POLGalpha. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGbeta is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.
Subject(s)
DNA, Mitochondrial/metabolism , DNA-Directed DNA Polymerase/metabolism , Protein Subunits/metabolism , Cell Line, Tumor , DNA Polymerase gamma , DNA, Mitochondrial/analysis , DNA, Mitochondrial/chemistry , DNA-Directed DNA Polymerase/genetics , Humans , Mitochondria/enzymology , Mitochondria/ultrastructure , Nucleic Acid Synthesis Inhibitors , Nucleoproteins/metabolism , Plasmids/chemistry , Protein Subunits/antagonists & inhibitors , Protein Subunits/genetics , RNA InterferenceABSTRACT
The nucleotide sequence of the Escherichia coli pgsA gene, encoding phosphatidylglycerophosphate synthase, is revised to code for an enzyme of 182 amino acid residues, instead of the 216 of a previous work (A. S. Gopalakrishnan, Y.-C. Chen, M. Temkin, and W. Dowhan, J. Biol. Chem. 261:1329-1338, 1986). The revised structure now explains the properties of the enzyme. Three pgsA mutants of different phenotypes were also analyzed: pgsA3, pgsA36, and pgsA10 have single-base replacements in codons 60 (Thr-->Pro), 1 (ATG-->ATA), and 92 (Thr-->Ile), respectively.
