Structure and Function of the Human Nuclear Pore Complex

The nuclear pore complex (NPC) mediates the selective transport of macromolecules between the nucleus and the cytoplasm of the eukaryotic cell. In humans, the NPC is a ~110 MDa assembly of ~1,000 proteins, termed nucleoporins, which establishes a ~50 nm wide central transport channel that traverses both membranes of the nuclear envelope. In the central transport channel, natively unfolded nucleoporin regions rich in repeated phenylalanine-glycine (FG) motifs form a size-selective barrier that limits the diffusion of macromolecules. Active cargo transport is facilitated by dedicated mobile transport factors that exchange through the FG barrier and couple binding and unbinding of cargo to GTP hydrolysis by the small GTPase Ran. By contrast, nuclear export of mature mRNA is driven by ATP-dependent remodeling of messenger ribonucleoprotein (mRNP) at the NPC's cytoplasmic face. Whereas the mechanisms of the mobile Ran dependent transport machinery have been conceptually rationalized, the NPC's role in facilitating and modulating nucleocytoplasmic transport, including mRNA export, remains poorly understood. Addressing gaps in the understanding of the NPC's structure and function, this thesis presents comprehensive interdisciplinary analyses of the NPC's inner ring, linker scaffold, and cytoplasmic face architectures. First, we determined the structure, stoichiometry, and location of the channel nucleoporin heterotrimer that provides the bulk of the FG repeats in the central transport channel. Second, we elucidated the molecular details and topology of the network of linker nucleoporins previously shown to assemble folded scaffold nucleoporins into protomeric subcomplexes of the inner ring. Third, we characterized the composition, stoichiometry, and attachment mechanism of cytoplasmic filament nucleoporins, which present manifold Ran- and RNA-binding domains on the cytoplasmic face of the NPC. The resulting near atomic structure of the human NPC provides a rich foundation for rationalizing the dilation and constriction of the central transport channel, the nucleocytoplasmic transport of integral membrane proteins, and the spatial arrangement of cytoplasmic filament nucleoporins that are involved in mRNA export and etiologies of several human diseases.