Comprehensive sub-mitochondrial protein map of the parasitic protist Trypanosoma brucei defines critical features of organellar biology

We have generated a high-confidence mitochondrial proteome (MitoTag) of the Trypanosoma brucei procy-clic 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 charac-teristic 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.

Automated summary

The generated MitoTag dataset provides new insights into the mitochondrial proteome of Trypanosoma brucei, identifying previously undescribed proteins and revealing sub-compartment localization and novel metabolic pathways.