This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The eukaryotic genome, and its associated proteins, is intricatelypackaged and sequestered within the boundary of a double membrane, knownas the nuclear envelope (NE). Transport across the NE is mediated by largeprotein assemblages known as nuclear pore complexes (NPCs). Yeast andvertebrate NPCs are comprised of about 30 proteins, termed nucleoporins(Nups), which are present in multiple copies. The origins and evolution of thenucleus and NPC are not yet clear, although it seems likely that the nucleusarose only once in eukaryotic evolution. To further our understanding of theevolution of the NPC, we characterized the NPC of a distantly related organism,relative to yeast and vertebrates. The parasitic protist Trypanosoma brucei is asuitable candidate for such a study due to its sequenced genome andexperimental tractability, compared to other protists. We have carried out acomprehensive analysis of the protein components of the trypanosome NPC.Towards this end, we used several biochemical and proteomic strategiesto identify the proteins that associate with a preparation of enriched T. bruceiNEs. Discerning authentic trypanosome Nups from the 859 proteins identifiedwas challenged by the large sequence divergence between yeast, vertebratesand trypanosomes. To overcome this challenge, we used a suite of rigorousbioinformatic tools, which allowed us to identify 24 putative Nups. In comparisonto the yeast and vertebrate NPC, we estimate that these putative Nups constituteat least 80%, by mass, of the trypanosome NPC. To further characterize theseputative Nups and the NPC, we employed RNAi. The results of these studiessuggest that, in addition to its role in nucleocytoplasmic transport, thetrypanosome NPC plays a key role in maintaining the stability and morphology ofthe NE.We then confirmed fully half of the putative trypanosome Nups byfluorescent localization, and observed that the density of trypanosome NPCsaround the nucleus is less than that of yeast or vertebrates. This lower densityenabled us to visualize individual NPCs and note differences in the spatialdistribution of NPCs between these three species.Despite significant divergence with respect to primary structure andspecies-specific innovations, the trypanosome NPC contains many homologs,domains and motifs found in opisthokonts. Given these findings, it is reasonableto infer that the architecture of the NPC is conserved across Eukaryota. Thissuggests that the NPC of the last common eukaryotic ancestor had manyfeatures in common with NPCs of contemporary bikonts (e.g. plants andexcavates) and opisthokonts (e.g. animals and fungi).
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