Metal-protein interactome in plant mitochondria

Yew-Foon Tan

Research output: ThesisDoctoral Thesis

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Abstract

[Truncated abstract] Transition metals in the plant mitochondrion have dual roles in regulating the function of the organelle. While metals participate in mitochondrial respiratory metabolism as ligands in bioenergetic, detoxifying, and various other metabolic enzymes, a breakdown in metal homeostasis during oxidative stress can perpetuate the cycling of ROS by redox active metal ions. Large-scale studies into the duplicitous roles of metal ions in biological systems has been lacking and in this thesis, a combination of metallomics, database annotations, membrane proteomics, metal-protein interactomics, structural biology, functional assays and mass spectrometry were all used to gain a clearer insight into the involvement of metal ions in affecting plant mitochondrial function. The Arabidopsis mitochondrion was shown to contain the transition metals cobalt, copper, iron, manganese, molybdenum, and zinc. Interestingly, the redox active copper and iron represented 75% of the mitochondrial metallome and these metal species were revealed to be highly labile during oxidative stress suggesting a possible contribution of metal-catalysed oxidation (MCO) in the damage of biological macromolecules. Bioinformatic analysis of metalloproteins predicted and experimentally determined to be mitochondrially localised revealed that metal ion transporters are poorly characterised. An in-depth proteomic analysis of the membrane proteome was conducted on mitochondria isolated from unstressed and stressed cell cultures resulted in the identification of stress-responsive as well as potential metal ion transporters. Also, many of the annotated metalloproteins predicted to be mitochondrial lack experimental evidence for subcellular localisation. ... However, based on evidence in the literature, it was hypothesised that metal-interacting sites may be the targets for MCO due to their affinity for metal ions. Attempts were made to identify the site specificity of MCO on mitochondrial proteins but no carbonyl sites could be found owing to technical problems associated with non-specific binding of proteins to the enrichment resin and low abundance of the labelled protein carbonyls. The use of the model protein BSA showed that protein oxidation occurs in clusters and the use of model peptides demonstrated that the ability of amino acid residues to complex metal ions is important in dictating susceptibility to MCO. Further experimental verification for the site specificity of MCO is required to determine the consequences of MCO on mitochondrial protein function. Overall, this thesis provided a large-scale analysis of the contributions of metal ions to mitochondrial respiratory metabolism with an emphasis on metal ion induced toxicity. Using multi-facetted approaches, an insight into the dynamic nature of mitochondrial metal homeostasis, stress responsive transporters, the interactions of metal ions with mitochondrial proteins and the possible mechanism in which proteins are specifically oxidised by MCO has been uncovered paving the way for future focused studies characterising the consequences of oxidative stress on specific proteins and their function.
Original languageEnglish
QualificationDoctor of Philosophy
Publication statusUnpublished - 2009

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