STRUCTURE AND FUNCTION OF THE NUCLEAR PORE COMPLEX Of central importance to the intracellular organization of all eukaryotes is the accurate transport of macromolecules between the nucleus and the cytoplasm, which is achieved through the function of the nuclear pore complex (NPC). The NPC is an enormous transport channel that perforates the double membrane of the nuclear envelope. However, how NPCs form and how they are inserted into the nuclear membrane is not known. NPCs play an essential role in many diverse transport events including the export of messenger RNAs from the nucleus to the cytoplasm. But despite its fundamental significance, the pathway by which messenger RNAs directionally translocate through the NPC remains poorly defined. The goals of this research proposal are to elucidate the mechanism by which NPCs form and assemble in the nuclear envelope and to characterize the role of the NPC in messenger RNA export. All experimental approaches described in this proposal take advantage of the proteomic, genomic and cell biological tools available for use within the single cellular eukaryote Saccharomyces cerevisiae. Yeast provides an excellent model system to characterize the components and function of the NPC, to study the complex NPC assembly process in living cells, and to develop novel reconstitution assays.
Our aims are: (1) To characterize the role of the NPC components Gle1 and Dbp5 in mRNA export and to investigate how mRNAs are unidirectionally transported across the NPC. (2) To study the role of the transmembrane nuclear pore protein Ndc1 in NPC assembly and structure. (3) To use fluorescence based assays to examine NPC biosynthesis in living cells and to reconstitute NPC assembly steps in vitro using defined components. We employ a combination of innovative biochemical, genetic and cell biological approaches to address these three specific aims in S. cerevisiae. Because the NPC is a highly conserved structure, the mechanistic insights obtained from these studies will be directly relevant to all eukaryotes, including humans.
In all eukaryotes, regulation of macromolecular transport through the nuclear pore complex provides an essential mechanism by which signal transduction pathways and developmental stimuli control differential gene expression. In addition, many viruses target components of the cellular nuclear transport machinery to facilitate viral propagation and several oncogenic translocations involve components of the nuclear pore complex to promote cancer development. Therefore, a better understanding of the molecular machinery that mediates nucleocytoplasmic transport is essential both for understanding fundamental cellular processes and the development of novel anti-viral and anti-cancer therapies.
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