Title: Proteomics of the Mitochondrion
Funder: Medical Research Council

Abstract: Proteomics of Mitochondria||We are using the technologies of Proteomics to identify, from more than 30,000 genes in the human genome, those important in cellular energy metabolism. The inner membrane of mitochondria is the site of cellular energy metabolism and contains the protein complexes of the respiratory chain as well as the ATP synthase and a family of metabolite transport proteins. Related processes include thermogenesis in brown fat mitochondria, proton leak, both major energy consumers, as well as cellular Ca++ regulation, maintenance of other cation gradients across the mitochondrial membrane, and the source of damaging oxygen radicals. More than 100 known proteins, comprise the protein complexes of the respiratory chain and ATP synthase. However, many of the inner membrane proteins that perform normal mitochondrial functions remain to be identified. Therefore we are using proteomic techniques to analyse the protein composition of the inner membrane of mitochondria so that novel components associated with energy metabolism can be identified. This involves the separation of proteins, often using electrophoresis methods, followed by protein identification, using the techniques of peptide mass fingerprinting and peptide sequence analysis with MALDI-TOF MS and tandem mass spectrometers respectively.||The characterisation of membrane proteins presents difficulties due to their hydrophobic nature and in mitochondrial membranes due also to their variability in abundance and the large number present. Whilst high-resolution separation techniques are required, many membrane proteins are too hydrophobic for separation by iso-electric focussing in conventional 2-D gels. When mitochondrial membrane components were resolved by simple 1-D electrophoresis and identified, by peptide mass fingerprinting and MALDI-TOF MS, only the more abundant mitochondrial proteins were identified. Most proteins were known components of membrane bound respiratory protein complexes. More rigorous purification strategies are required for the characterisation of rare and unknown proteins such as low abundance ion channels and proton and metabolite transporters. Methods for the purification of membrane proteins and their analysis by mass spectrometry are currently underdeveloped and suitable strategies are being investigated. Protein complexes from mitochondrial membranes, with relatively simple subunit compositions are also being characterised. We have defined the subunit composition of Complex I and in particular smaller sub-complexes from both mitochondrial and bacterial sources. These analyses are in support of the structural investigations undertaken by the research groups of Drs Hirst and Sazanov in the Mitochondrial Biology Unit. Protein complexes associated with replication of mitochondrial DNA, known as the nucleoid, are being isolated and analysed in collaboration with Dr I Holt (MBU). Several significant new components have been identified and their functions are being studied. The ATP synthase complex is being examined for evidence of modification by phosphorylation, involved in the regulation of its activity. In addition, the functions of a group of mitochondrial membrane proteins are being characterised. Two of them are loosely associated with the ATP synthase complex.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE