Nuclear pore complexes (NPCs) are large proteinaceous assemblies that provide the only known portals for the exchange of macromolecules between the nucleus and cytoplasm. Revealing how the transport apparatus is assembled will be critical to understanding the mechanism of nucleocytoplasmic communication. The goal of our work is to investigate the pathway of NPC biogenesis at the molecular level. Our experimental method is designed to benefit from the strength of combining molecular genetic, biochemical, and cell biological approaches. Our first specific aim is to identify genes that regulate NPC formation with a fluorescence-based screen in the yeast Saccharomyces cervisiae. This approach is based on the functional tagging of NPC proteins with the green-fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP signals as compared to wild type cells. These GFP signal differences will allow mutants to be isolated by fluorescence activated cell sorting and direct microscopic screening. Three classes of mutants are expected: 1) NPC clustering, 2) fewer total NPCs per nucleus, and 3) wild type NPC number, but each NPC has a decreased amount of GFP-protein incorporated. In the second aim, we will analyze the mechanism of action of potential assembly factors by assaying the mutants in situ for perturbations of NPC assembly. NPC density and general morphological structure analysis will be assessed by electron microscopy. Rate of NPC assembly will be determined by two complementary approaches: monitoring incorporation of GFP-proteins in live cell assays, and pulse chase analysis. NPC dynamics will be monitored with a live cell assay measuring NPC movement rates. This analysis will include both novel mutants and a nup57 mutant identified in our preliminary studies.
We aim to reveal whether the proteins are required for the incorporation of a nearest neighbor interacting protein(s) or if they are global assembly factors required for multiple aspects of NPC structure. The third specific aim is focused on characterizing a novel integral membrane protein with connections to NPC function. We will determine the membrane topology of Sn11p, and test for genetic and biochemical interactions with NPC components. Finally, we propose to isolate vertebrate homologues of yeast NPC assembly factors, and conduct direct tests for their roles in NPC assembly with the established Xenopus nuclear assembly assay. Together these studies are expected to significantly advance our understanding of how the nuclear transport apparatus is assembled and functions.
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