The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied in Saccharomyces cerevisiae. Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA. However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm. In this article we have used immunocytochemistry and biochemical isolation procedures to characterize the composition of human mitochondrial nucleoids. The results show that both the mitochondrial transcription factor TFAM and mitochondrial single-stranded DNA-binding protein colocalize with Twinkle in intramitochondrial foci defined as nucleoids by the specific incorporation of bromodeoxyuridine. Furthermore, mtDNA polymerase POLG and various other as yet unidentified proteins copurify with mtDNA nucleoids using a biochemical isolation procedure, as does TFAM. The results demonstrated that mtDNA in mammalian cells is organized in discrete protein-rich structures within the mitochondrial network. In vivo time-lapse imaging of nucleoids show they are dynamic structures able to divide and redistribute in the mitochondrial network and suggest that nucleoids are the mitochondrial units of inheritance. Nucleoids did not colocalize with dynaminrelated protein 1, Drp1, a protein of the mitochondrial fission machinery. INTRODUCTIONMammalian mitochondrial DNA (mtDNA) is a 16.5-kb circular double-stranded DNA (Anderson et al., 1981;Bibb et al., 1981) present in one to several thousand of copies per cell (e.g., Takamatsu et al., 2002). All proteins involved in mtDNA maintenance are encoded by the nuclear genome. These are traditional proteins in replication and repair such as the mtDNA polymerase POLG, but also include proteins directly or indirectly involved in e.g., segregation.MtDNA in the yeast Saccharomyces cerevisiae and to a lesser extent in a few other species, appears to be organized in discrete foci within mitochondria called nucleoids (Miyakawa et al., 1984). These have been inferred to be the units of inheritance each containing several copies of yeast mtDNA MacAlpine et al., 2000 and references therein). Biochemical purification and protein analysis have defined several of the constituents of yeast nucleoids (Miyakawa et al., 1995;Newman et al., 1996;Kaufman et al., 2000). One of the core components is the Abf2 protein (Abf2p), an orthologue of the human mitochondrial transcription factor TFAM. The function of Abf2p in yeast is essential for mtDNA maintenance by providing a mtDNApackaging function. It also modestly stimulates yeast transcription in in vitro assays (Parisi et al., 1993). A mouse TFAM knockout shows embryonic lethality with complete loss of mtDNA (Larsson et al., 1998), but this is generally believed to be the result of impairment of transcription initiation that would generate primers for mtDNA replication.Other yeast nucleoid components include Rim1p, the yeast s...

Tid1 is a human homolog of bacterial DnaJ and the Drosophila tumor suppressor Tid56 that has two alternatively spliced isoforms, Tid1-long and -short (Tid1-L and -S), which differ only at their carboxyl termini. Although Tid1 proteins localize overwhelmingly to mitochondria, published data demonstrate principally nonmitochondrial protein interactions and activities. This study was undertaken to determine whether Tid1 proteins function as mitochondrial DnaJ-like chaperones and to resolve the paradox of how proteins targeted primarily to mitochondria function in nonmitochondrial pathways. Here we demonstrate that Tid1 isoforms exhibit a conserved mitochondrial DnaJ-like function substituting for the yeast mitochondrial DnaJ-like protein Mdj1p. Like Mdj1p, Tid1 localizes to human mitochondrial nucleoids, which are large protein complexes bound to mitochondrial DNA. Unlike other DnaJs, Tid1-L and -S form heterocomplexes; both unassembled and complexed Tid1 are observed in human cells. Results demonstrate that Tid1-L has a longer residency time in the cytosol prior to mitochondrial import as compared with Tid1-S; Tid1-L is also significantly more stable in the cytosol than Tid1-S, which is rapidly degraded. The longer cytosolic residency time and the half-life of Tid1-L are explained by its interaction with cytosolic Hsc70 and potential protein substrates such as the STAT1 and STAT3 transcription factors. We show that the unique carboxyl terminus of Tid1-L is required for interaction with Hsc70 and STAT1 and -3. We propose that the association of Tid1 with chaperones and/or protein substrates in the cytosol provides a mechanism for the alternate fates and functions of Tid1 in mitochondrial and nonmitochondrial pathways.Tid1 is a human homolog of bacterial DnaJ and the Tid56 tumor suppressor of Drosophila melanogaster. DnaJ-like proteins function as co-chaperones with DnaK-like ATPases to promote the folding, translocation, and/or degradation of polypeptides (1-3). DnaJ-and DnaKlike proteins are highly conserved, and homologs are found in nearly every compartment of the eukaryotic cell and in several tumor viruses (4). In Drosophila, the absence of Tid56 results in abnormal differentiation and morphogenesis, giving rise to Tumorous imaginal discs that lead to lethality during early development (5). The mechanism underlying Tid56 function is unknown.In humans, two alternatively spliced forms of Tid1 are expressed, Tid1-long (Tid1-L) and Tid1-short (Tid1-S), that differ only at their carboxyl-terminal tails (Fig. 1A) (6). Tid1-L contains 33 amino acids unique to its carboxyl terminus, whereas Tid1-S has 6 amino acids (Fig. 1A). Both Tid1-L and -S have a predicted amino-terminal mitochondrial targeting sequence as well as the signature J-domain carrying an HPD motif required for stimulating the ATPase activity of eukaryotic DnaK-like chaperones (1-3). Experiments in mice show that Tid1 is critical for early mammalian development (7). Mice specifically deficient in Tid1 in the heart develop dilated cardiomyopathy, progre...

Cisplatin accumulates in mitochondria, which are a major target for this drug in cancer cells. Thus alterations in mitochondrial function have been implicated in cancer cell resistance to chemotherapeutic agents. Moreover, cisplatin toxic side effects seem to be associated with mitochondrial injury in vivo and in vitro. In order to clarify the potential effect of cisplatin in mtDNA (mitochondrial DNA) maintenance and expression, we have analysed rat liver mtDNA and mtRNA (mitochondrial RNA) synthesis as well as their stability under the influence of in vivo treatment or in vitro exposure to cisplatin. We show that cisplatin causes a direct and significant impairment of mtDNA and mtRNA synthesis and decreases steady-state levels of mtRNAs in isolated mitochondria. Furthermore, in vivo treatment of the animals with cisplatin exerts a protective effect from the impairment of mtRNA metabolism caused by in vitro exposure to the drug, by means of increased mitochondrial GSH levels after in vivo cisplatin treatment.