We have generated a high-confidence mitochondrial proteome (MitoTag) of the Trypanosoma brucei procyclic stage containing 1,239 proteins. For 337 of these, a mitochondrial localization had not been described before. We use the TrypTag dataset as a foundation and take advantage of the properties of the fluorescent protein tag that causes aberrant but fortuitous accumulation of tagged matrix and inner membrane proteins near the kinetoplast (mitochondrial DNA). Combined with transmembrane domain predictions, this characteristic allowed categorization of 1,053 proteins into mitochondrial sub-compartments, the detection of unique matrix-localized fucose and methionine synthesis, and the identification of new kinetoplast proteins, which showed kinetoplast-linked pyrimidine synthesis. Moreover, disruption of targeting signals by tagging allowed mapping of the mode of protein targeting to these sub-compartments, identifying a set of C-tail anchored outer mitochondrial membrane proteins and mitochondrial carriers likely employing multiple target peptides. This dataset represents a comprehensive, updated mapping of the mitochondrion.

Division of Biomedical Sciences Warwick Medical School University of Warwick Coventry UK

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic; Department of Biochemistry University of Cambridge Cambridge UK; Faculty of Science University of Ostrava Ostrava Czech Republic

Oxford Brookes University Oxford UK

Peter Medawar Building for Pathogen Research Nuffield Department of Medicine University of Oxford Oxford UK

Sir William Dunn School of Pathology University of Oxford Oxford UK

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