Structural and biophysical characterization of membrane-coating proteins from the nuclear pore and the primary cilium

Abstract

A hallmark of eukaryotes is an endomembrane system that spatially separates cellular processes into discrete compartments. Macromolecular transport between these compartments canonically involves the fission and fusion of membrane-bound vesicles. Transport to and from the nucleus is a notable exception to this vesicular pathway. Additionally, transport to the primary cilium is not well characterized and is therefore of interest. The nuclear envelope comprises a double membrane that fuses at points to produce pores, which link the nucleoplasm with the cytoplasm. Coating these openings are nuclear pore complexes (NPCs), which regulate all nucleocytoplasmic trafficking by anchoring barrier forming FG-repeat proteins. At 60-120 MDa, the NPC is the largest macromolecular complex in the cell. However, it is a modular assembly formed by -30 different nucleoporins (Nups) arranged into stable subassemblies. One such module is the -600 kDa Y complex, which forms a Y shape and is the best characterized NPC subcomplex with crystal structures accounting for -90% of its total mass. The molecular details of how the short arms of the Y contact the long stem, in a region called the 'hub', were not known. We solved the structure of this last major missing piece of the Y complex to 4.1 A. This hub structure revealed unexpected curvature, allowed us to build the first atomic resolution composite of the mostly complete Y, and led to a novel higher order assembly model of the Y complex in the intact NPC. The ciliary membrane is topologically contiguous with the plasma membrane yet functionally distinct, due to a unique complement of integral membrane proteins. The Bardet- Biedl syndrome protein complex (BBSome) functions in the establishment or maintenance of this unique composition. The BBSome is -500 kDa octamer comprising BBS1, 2, 4, 5, 7, 8, 9, and BBIP10. To date, only the N-terminal domain of BBS1 had been structurally determined. In order to more completely characterize this complex, we have solved the structure of the BBS9 N-terminal [beta]-propeller to 1.8 A and determined its oligomeric state in solution. This structure has allowed us to identify a putative interaction site and characterized a disease relevant mutation on BBS9.