Comparative Proteomics: Studies on the Composition and Evolution of the Mitochondrial Proteome in Eukaryotic Microbes (Protists).
Supplemental Figure 2.2 In-gel cytochrome c oxidase and ATPase enzyme assays of A. castellanii mitochondria solubilized with different amounts of DDM (final concentrations depicted for each lane by “%”). (8.649Mb)
Supplemental Figure 3.1. Phylogenetically broad alignment of eukaryotic and prokaryotic γCAs. (60Kb)
Supplemental Figure 4.1. Phylogenetically broad alignment of the N-terminal portion of SdhB. (53.16Kb)
Supplemental Figure 4.2. Phylogenetically broad alignment of the C-terminal portion of SdhB. (52.36Kb)
Supplemental Figure 4.3. Maximum likelihood phylogenetic tree of concatenated SdhB-n and SdhB-c proteins. (14.81Kb)
Supplemental Table 4.1. Database accession numbers, annotation, gene location, protein size. (10.5Kb)
MetadataShow full item record
Mitochondria are eukaryotic organelles derived in evolution from within the ? subdivision of Proteobacteria. Although mitochondria are structurally and metabolically complex, modern-day mitochondrial genomes (mtDNA) encode only a small number of RNAs and proteins predominantly involved in adenosine triphosphate (ATP) formation through electron transport coupled to oxidative phosphorylation, as well as translation of mtDNA-encoded proteins. In humans, only 13 of the >1000 polypeptides that constitute the complete mitochondrial protein complement (proteome) are encoded in mtDNA; the remainder is encoded by nuclear DNA (nuDNA). It is therefore imperative to comprehensively catalog nuDNA-encoded mitochondrial proteins in order to understand holistically the evolution of mitochondria. Mitochondrial proteome investigations of animals, fungi and land plants have dramatically altered our conception of mitochondrial evolution: in contrast to mtDNA-encoded proteins, few nuDNA-encoded mitochondrial proteins are demonstrably derived from the eubacterial progenitor of mitochondria, and many are found only in eukaryotes. Notably, however, little is known about the mitochondria of eukaryotic microbes (protists), which constitute the bulk of biochemical and genetic diversity within the domain Eucarya. The proteomic characterization of protist mitochondria is therefore crucial to fully elucidating mitochondrial function and evolution. Employing tandem mass spectrometry (MS/MS), I have analyzed highly purified mitochondria from Acanthamoeba castellanii (Amoebozoa). In combination, nearly 750 nuDNA- and mtDNA-encoded proteins were identified. These data were used to catalog metabolic pathways and protein complexes, and to infer functional and evolutionary profiles of A. castellanii mitochondria. My analyses suggest that while A. castellanii mitochondria have many features in common with other eukaryotes, they possess several novel attributes and pronounced metabolic versatility. An analysis of the A. castellanii electron transport chain (ETC) was also performed, utilizing a combination of blue native polyacrylamide gel electrophoresis (BN-PAGE), MS/MS and bioinformatic queries. A significant proportion of A. castellanii ETC proteins was identified, yielding several insights into ETC evolution in eukaryotes. Lastly, I present two unusual cases of ‘split’ mitochondrial proteins: the iron-sulfur subunit SdhB of succinate:ubiquinone oxidoreductase (Complex II), in the phylum Euglenozoa and Cox1 of cytochrome c:O2 oxidoreductase (Complex IV) in various eukaryotes, including A. castellanii. Functional and evolutionary implications of these findings are discussed.