Proteomics of the Mitochondrion

Lead Research Organisation: Medical Research Council

Abstract

Mitochondria are organelles within all cells responsible for the oxidation of sugars from derived from food and the conversion of that energy into ATP, the form of energy used at a cellular level. The energy conversion processes occur in the mitochondrial membranes and are catalysed by a series of respiratory protein complexes. We are analysing the protein content of these mitochondrial membranes to discover new proteins that contribute to energy transduction.

Technical Summary

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.

Publications

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Andrews B (2013) Assembly factors for the membrane arm of human complex I. in Proceedings of the National Academy of Sciences of the United States of America

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Carroll J (2013) Post-translational modifications near the quinone binding site of mammalian complex I. in The Journal of biological chemistry

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Carroll J (2006) Bovine complex I is a complex of 45 different subunits. in The Journal of biological chemistry

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Carroll J (2006) Definition of the mitochondrial proteome by measurement of molecular masses of membrane proteins. in Proceedings of the National Academy of Sciences of the United States of America

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Carroll J (2007) Identification of membrane proteins by tandem mass spectrometry of protein ions. in Proceedings of the National Academy of Sciences of the United States of America

 
Title Development of mass spectrometric methods for analysis of membrane proteins. 
Description Development of mass spectrometric methods for analysis of membrane proteins. Extraction of hydrophobic proteins in organic solvents, chromatographic methods to fractionate the hydrophobic proteins in the extracts, procdures for determining sequences in the proteins by mass spectrometry 
Type Of Material Improvements to research infrastructure 
Year Produced 2008 
Provided To Others? Yes  
Impact Discovery of new membrane proteins in mitochondria. Characterization of the thirteen proteins that are encoded in human mitochondrial DNA. 
 
Description Mitochondria from omental fat 
Organisation University of Basel
Country Switzerland 
Sector Academic/University 
PI Contribution We have compared the proteomes of mitochondria isolated from omental fat and from "normal" adipose tissue, and identified significant differences. We have purified respiratory enzyme complexes from the omental fat mitochondria and compared their subunit compositions.
Collaborator Contribution joint publication
Impact The experiments to characterize the respiratory capabilities of the omental fat mitochondria are being studied by our collaborators. Once they have been completed a manuscript will be prepared for publication.
 
Description Mitochondrial sirtuins 
Organisation National Institutes of Health (NIH)
Country United States 
Sector Public 
PI Contribution We have been studying the acetylation of mitochondrial proteins by the sirtuin, SirT3. One protein that is acetylated is the oscp subunit of the ATP synthase complex. The site of acetylation is being investigated.
Collaborator Contribution Identification of subunits ofn ATPase transiently modified by acetylation
Impact The work is being completed at the NIH and a publication is being planned.
Start Year 2007
 
Description Studies of PINK1 in relation to Parkinson's disease 
Organisation National Institutes of Health (NIH)
Department National Institute of Neurological Disorders and Stroke (NINDS)
Country United States 
Sector Public 
PI Contribution Protein interactions with PINK
Collaborator Contribution Role of PINK in mitophagy
Impact Publication and another in preparation PhD thesis
Start Year 2008
 
Description Publications 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Various conferences, named lectures, visits to sixth form colleges

Greater insight into the field
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013