Nuclear pore complexes (NPCs) provide the sole gateways that control the bidirectional exchange of molecules across the nuclear envelope (NE) in all eukaryotes. From the perspective of human health, compromised function of several of the components of the NPC (nucleoporins/nups) is associated with diverse diseases including cancers, heart disease, triple A syndrome, and neurodegenerative diseases like Alzheimer's and Parkinson's. Further, to propagate and promote infection, viruses often modulate nup function. The wide spectrum of these pathologies suggests that the NPC impacts a broad array of essential cellular processes, although the mechanisms are poorly defined. In addition, several nup genes are up- or down- regulated, and NPC number is altered, in developmental and disease contexts. Thus, a better understanding of mechanisms that contribute to NPC function will likely reveal translational drug targets in the future. While, as a field, we have a good understanding of the underlying mechanisms governing nuclear transport, a major remaining challenge is determining the mechanism of de novo NPC assembly. Specifically, it is not understood how the ~30 individual nups are coordinately assembled in space and time to form the ~50 MD NPC. Further, the assembly of individual nups coincides with the generation of membrane curvature that leads to the close apposition and eventual fusion of the inner and outer nuclear membranes to generate a nuclear pore. It is not known how nup assembly and membrane fusion are coordinated, nor has the fusion machinery been clearly identified. In this proposal, we aim to tackle two key challenges in understanding the assembly of the NPC. First, we propose an experimental strategy designed to elucidate the order by which nups are assembled at the NE during interphase. We will achieve this by exploiting the genetic toolkit of the yeast, S. cerevisiae, to generate a system where we rapidly and specifically inactivate newly synthesized nups one at a time, leaving mature NPCs unaffected. After inactivation of each target nup, we will comprehensively examine the distribution of other nups to assign an up- or down-stream relationship in the order of assembly. Integrating this data set, we will systematically define the steps in the assembly process. Second, we will use both the order analysis and a candidate approach to identify proteins that generate membrane curvature to support pore formation in the NE during NPC assembly. Using a series of in vivo and in vitro approaches, we will test whether these candidate curvature generators are capable of directly driving membrane curvature and home in on their curvature-generating domains. In this way, we will shed significant new light onto the molecular mechanisms of pore formation and directly test how membrane curvature impacts NPC assembly. The experiments outlined in this proposal will provide much needed mechanistic insight into the essential process of NPC biogenesis, universal to all eukaryotes.
Nuclear pore complexes are massive protein gateways that control the compartmentalization of the nucleus and cytoplasm. Understanding how nuclear pore complexes are assembled will provide new insight into how their number, composition and function are altered in developmental and disease- specific contexts and will suggest new targets in therapeutic strategies.
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