Membrane translocases in a mitochondrially derived organelle: a study of the T. vaginalis hydrogenosome

Eukaryote cells are the products of a complex history of interspecies interactions, with some organelles now known to have arisen through the endosymbiosis of prokaryotic cells. Whilst these organelles 'evolved' from the original endosymbionts, their evolution has not stopped within the modern eukaryote. Trichomonas vaginalis is protozoan parasite, with an unusual cellular biology, this species appears to lack peroxisomes, and instead of mitochondria has divergent organelles called hydrogenosomes. The hydrogenosomes of Trichomonas represent one of a growing number of highly divergent organelles, which are present in species throughout the eukaryotic kingdom. In this investigation the hydrogenosomes of Trichomonas are investigated with respect to their preprotein membrane translocases, a multi-membraned molecular system essential for the organelle's maintenance and biogenesis. The complexity and components of such a system are unlikely to arise duplicated from a separate organellogenesis, and thus the system's architecture is expected to indicate this organelle's descent. To reveal the structure of this system two different practical approaches were used to determine the biology of the hydrogenosome. The first builds upon work to characterise translocase kinetics and probes the nature of the hydrogenosomal membrane translocon directly. The second explores the use of candidate translocases determined from the Trichomonas genome through bioinformatic analysis and their development into practical investigation through the expression of tagged proteins in transformant T. vaginalis. These transformants were used to visualise a population of membrane proteins in situ within the hydrogenosome by immunofluorescence microscopy, and further to identify their associations and interactions within the hydrogenosomal membrane using protein biochemical methods. The data produced within this study are finally brought together to present a model for the Trichomonas hydrogenosomal preprotein import system, as well as the first molecular characterization of its translocase components