Date of Award
nuclear pore complex, trypanosoma brucei
The eukaryotic genome, and its associated proteins, is intricately packaged and sequestered within the boundary of a double membrane, known as the nuclear envelope (NE). Transport across the NE is mediated by large protein assemblages known as nuclear pore complexes (NPCs). Yeast and vertebrate NPCs are comprised of about 30 proteins, termed nucleoporins (Nups), which are present in multiple copies. The origins and evolution of the nucleus and NPC are not yet clear, although it seems likely that the nucleus arose only once in eukaryotic evolution. To further our understanding of the evolution of the NPC, we characterized the NPC of a distantly related organism, relative to yeast and vertebrates. The parasitic protist Trypanosoma brucei is a suitable candidate for such a study due to its sequenced genome and experimental tractability, compared to other protists. In this thesis, we present the comprehensive analysis of the protein components of the trypanosome NPC. Towards this end, we used several biochemical and proteomic strategies to identify the proteins that associate with a preparation of enriched T. brucei NEs. Discerning authentic trypanosome Nups from the 859 proteins identified was challenged by the large sequence divergence between yeast, vertebrates and trypanosomes. To overcome this challenge, we used a suite of rigorous bioinformatic tools, which allowed us to identify 24 putative Nups. We then confirmed fully half of the putative trypanosome Nups by fluorescent localization, and observed that the density of trypanosome NPCs around the nucleus is less than that of yeast or vertebrates. This lower density enabled us to visualize individual NPCs and note differences in the spatial distribution of NPCs between these three species. To further characterize these putative Nups and the NPC, we employed RNAi. The results of these studies suggest that, in addition to its role in nucleocytoplasmic transport, the trypanosome NPC plays a key role in maintaining the stability and morphology of the NE. Despite significant divergence with respect to primary structure and species-specific innovations, the trypanosome NPC contains many homologs, domains and motifs found in opisthokonts. Given these findings, it is reasonable to infer that the architecture of the NPC is conserved across Eukaryota. This suggests that the NPC of the last common eukaryotic ancestor had many features in common with NPCs of contemporary bikonts (e.g. plants and excavates) and opisthokonts (e.g. animals and fungi).
DeGrasse, Jeffrey Allen, "Proteomic, Bioinformatic and Functional Characterization of the Nuclear Pore Complex of the African Trypanosome" (2008). Student Theses and Dissertations. 26.