Structural and biochemical analysis of the Y-shaped Nup84 subcomplex of the NPC

The eukaryotic cell is easily distinguishable from its prokaryotic counterpart through the presence of a complex endomembrane system. Most notable is the nucleus, which harbors and protects the genetic information of the cell. Such physical separation allows for extra layers of regulation for a variety of cellular processes, but at the same time, necessitates a mechanism for communication between the nucleus and cytoplasm. The nuclear envelope consists of an inner and outer nuclear membrane that fuse at distinct loci to form a gateway into the nucleus. Embedded into these circular openings are nuclear pore complexes (NPCs), which serve as the gatekeepers, mediating all exchange between the nucleus and the cytoplasm. At its core, the NPC consists of an 8-fold symmetric structural scaffold that serves as a docking site for some of the more dynamic components. Overall, the NPC is composed of -30 proteins, or nucleoporins (nups), that form biochemically stable subcomplexes which are repeated in multiple copies to assemble the intact NPC. This macromolecular machine is not only essential to nucleo-cytoplasmic transport, but also plays a pivotal role in a myriad of other cellular processes. Thus, understanding its function and assembly in molecular detail is of great interest and importance. Here, I investigate the structure and assembly of one of the major components of the NPC scaffold, the Nup84 complex. Also known as the Y complex, this 7- membered assembly adopts the shape of an extended Y in solution. We solved the structure of the heterodimeric Nup85 Sehl complex, which forms one of the short arms of the Y. Nup85 was found to have an unexpected, yet conserved fold, termed ancestral coatomer element 1 (ACEI), which is also present in 3 additional scaffold nucleoporins, as well as Sec3l, a major component of COPIl vesicle coats. This discovery led to the first experimental evidence for a common ancestry of nucleoporins and vesicle coat proteins. Additionally, we solved the partial structure of Nup120, which also exhibited a unique and unexpected domain architecture. While initial secondary structure prediction methods classified all nups into the canonical a-solenoid and p-propeller fold types, an arsenal of recent nucleoporin structures now tells a different story. To date, at least a partial structure of each of the components of the Nup84 complex has been solved. Using biochemical interaction data as a guide, we can now place each of these structures into an electron density map generated by electron microscopy (EM) to build an initial composite structure of a nearly complete Y-shaped Nup84 complex. Furthermore, we developed the lattice model for assembly of the NPC structural scaffold based on the similarities discovered between nucleoporins and COPII vesicle coats-two membrane-coating complexes whose proteins are evolved from a common ancestor. To develop the lattice model further, deciphering the complex interaction network linking each of the biochemically-defined subcomplexes will be paramount in arriving at a more detailed and accurate model that can provide mechanistic insight into NPC function.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2012

Author dc:creator

• Leksa, Nina Carolina

Advisor dc:contributor.advisor

• Thomas U. Schwartz.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/70390

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/70390