Structural and Functional Characterization of theConserved Nup82 Subcomplex Located on theCytoplasmic Side of the Yeast Nuclear Pore Complex

Abstract

Nuclear pore complexes (NPCs) are huge, proteinaceous assemblies, embedded in the nuclear envelope that act as gateways between the cytoplasm and nucleoplasm. A single NPC is formed from ~30 different nucleoporins, present in multiple copies. The vast majority of nucleoporins is organized in distinct subcomplexes. A number of NPC modules have been described in the past, but the structure of the tetrameric Nup82-Nup159C-Nsp1C-Dyn2 complex, located at the cytoplasmic side of the NPC and involved in nuclear mRNA export, remained elusive. My PhD study has focused on the conserved C-domain of Nup159, the key subunit of the Nup82 complex. Nup159-C harbors an essential -helical subdomain (H1), which is able to homo-dimerize through a coiled-coil interaction. Mutation in this region disturbs homo-dimerization and Nup82 complex assembly. The linker sequence prior to the essential H1 sub-domain turned out to be critical for dynein light chain (Dyn2) recruitment at the DIDNup159 motif (dynein light chain interacting domain), which binds five Dyn2 dimers. This finding suggests that DIDNup159 and H1 domains require a certain distance for Dyn2 binding to ensure the efficient Nup82 complex assembly and incorporation into the NPC scaffold. To gain more insight into the structure of the Nup82 complex, I have applied a method to reconstitute in vivo and subsequently isolate the essential structural core of the Nup82 module devoid of flexible FG repeats. Biochemical, biophysical and electron microscopy analysis unraveled that the Nup82 complex consists of a 20 nm stalk formed by the DIDNup159-Dyn2 and an asymmetric ’head’ structure that contains four Nup82 molecules with different properties and the  helical C-domains of Nup159 and Nsp1, leading to a large assembly with a molecular mass in the megadalton range. Furthermore, cross-linking mass spectrometry allowed to map the interaction regions between four subunits of the complex. In conclusion, the study revealed for the first time the three dimensional (3D) architecture of the Nup82-Nup159C-Nsp1C-Dyn2 complex with Nup159 C-domain forming the key self-dimerizing scaffold.