The Architecture of the Nuclear Pore Complex

Abstract

Nucleocytoplasmic transport, the regulated trafficking of macromolecules in and out of the nucleus, occurs primarily through nuclear pore complexes (NPCs). NPCs are massive macromolecular machines embedded in the nuclear envelope which generate ~40 nanometer transport channels to facilitate transport. Because of its size and complexity (~1000 subunits, ~120 MDa), the structure of the NPC has remained poorly understood. This thesis presents a bottom-up approach to understanding the structure and function of the NPC through reconstitution of the proteins and structural and biochemical studies. The first three chapters present work towards determining the composite structure of the symmetric core of the NPC. X-ray crystal structures are described for many of the components of the symmetric core. This includes a heterohexameric coat nucleoporin complex containing Nup120, Nup85, Nup145C, Sec13, Seh1, and Nup84, revealing how these proteins assemble into one of the main subcomplexes in the NPC. Reconstitution of the symmetric core components and analysis of the protein-protein interaction between the components provides a detailed biochemical map for the protein interaction network in the NPC. X-ray crystal structures of overlapping fragments facilitate the generation of accurate atomic model for full-length proteins. An iterative, sequential docking approach is developed to dock these models into a cryoelectron tomographic reconstruction of the human NPC, yielding a composite model for the structure of the symmetric core of the NPC. In the next two chapters, this analysis is extended to the cytoplasmic-specific decorations of the NPC. The structure of the C-terminal domain of Nup358 is reported and its catalytic activity is described. Lastly, reconstitution of human DDX19 activation by the NPC reveals mechanistic insight into how the NPC directly regulates the last step of mRNA export.