Dissecting the Mammalian Mitochondrial Nucleoid

Most of the body's energy comes from food which is converted to ATP, the energy currency of the cell. Respiration is the most efficient means of making ATP, a process known as oxidative phosphorylation, and this takes place in a compartment of the cell called mitochondria. Oxidative phosphorylation requires five multisubunit protein complexes. The vast majority of DNA in the cell is contained in the nucleus; however, 13 proteins are produced from DNA in mitochondria, so-called mitochondrial DNA. Unfortunately these 13 proteins are not trivial, but essential to life. They represent key components of the oxidative phosphorylation system. Defects in mitochondrial DNA cause a wide range of diseases in humans and there is growing evidence that they contribute to the natural process of ageing. Mitochondrial DNA, like every other DNA, requires a host of proteins to ensure its faithful reproduction, less obviously it also requires proteins for its organisation, maintenance and segregation. Thus, the depiction of mitochondrial DNA, as an open circle floating free in the mitochondrial matrix without protein, in many textbooks is erroneous. In reality, mitochondrial DNA is organized in multi-genomic nucleoprotein complexes, or nucleoids. The inventory of proteins associated with yeast mitochondrial DNA is closer to completion than that of higher eukaryotes (including humans), however, it is clear that there are substantial differences in the protein composition of animal and yeast mitochondria nucleoids (Chen & Butow 2005). Recently, we have identified a number of new proteins that associate with mitochondrial DNA, using a generic DNA binding protein to capture mitochondrial nucleoprotein complexes. This has opened up a new area of biology and we now plan to characterise these proteins in detail, in order to understand how mammals maintain their mitochondrial DNA and ensure its successful transmission to offspring. Reference: Chen, X.J. and Butow, R.A. (2005) The organization and inheritance of the mitochondrial genome. Nat Rev Genet, 6, 815-25.

The isolation of mitochondrial nucleoprotein complexes led to the identification of a novel DNA binding protein, ATAD3p (He et al., 2007). A portion of ATAD3p binds preferentially to DNA with a D-loop; this was a provocative finding as many molecules of mtDNA contain a triple-stranded region or D-loop, which hitherto had no known function, despite being characterised over 30 years ago. Gene silencing of ATAD3 alters the structure of mitochondrial nucleoids and causes extracted mtDNA multimers to fall apart, leading us to propose that the protein binds to mitochondrial D-loops and contributes to mtDNA formation and organisation. There is still much that we do not understand about ATAD3p, for instance the function of its AAA domain is opaque. Therefore, we plan to elucidate further details of the properties of ATAD3p, with the aim of understanding its function in mtDNA metabolism. We will ablate the ATPase activity of the protein by site-directed mutagenesis and study the effects of the mutations on the properties of the protein in cultured cells. Other new candidates will also be investigated, and the original purification method will be refined with the aim of yielding new proteins that interact with mitochondrial DNA. Based on the complexity of bacterial nucleoids, it is highly likely that other players remain to be unearthed. Defining the proteome of mitochondrial nucleoids is an essential antecedent to understanding their purpose. As the project progresses we will begin to study nucleoids in toto, seeking to address such issues as the role of nucleoids in regulating mtDNA copy number, and in limiting free radical damage, or ameliorating its effects via gene conversion. Testing such hypotheses is fraught without an accurate inventory of the mitochondrial nucleoid and a clear understanding of the function of its component parts. Reference: He, J., et al. (2007) J Cell Biol, 176, 141-6.