The nucleus is the inner sanctuary of the eukaryotic cell, keeping the genetic information enclosed by the nuclear envelope (NE). The NE consists of two concentric membrane bilayers fused at specific sites to form circular openings occupied by the nuclear pore complex (NPC). As the only gateway into and out of the nucleus, the NPC plays a critical role in cellular homeostasis, but it is more than a transport organelle. We are beginning to appreciate that the NPC also serves as a reference point to organize the nucleus and is likely to play a pivotal role in gene regulation. This research program focuses on determining the molecular architecture of the enormous ~50 MDa NPC. Attaining this goal will provide a structural basis to interrogate its myriad functions in transport, gene regulation, and nuclear organization. Importantly, aberrant function of the NPC and its constituents is a primary cause of many human diseases, including leukemia, other cancers, autoimmune diseases, cardiomyopathies, and a variety of viral infections. The NPC is a modular structure, composed of ~30 different proteins, so-called nucleoporins that arrange in multiple copies around a central eightfold rotational axis. Subcomplexes of 2-10 nucleoporins hierarchically build up the NPC. Two of these subcomplexes, the heteromeric Y- and Nic96-complexes, organize the majority of architectural nucleoporins and establish the stable scaffold of the NPC. Consequently, these two subcomplexes are the main focus of this proposal. Once both are fully structurally characterized, the scaffold structure of the NPC can likely be generated with high confidence. This proposal is organized around four aims.
Aim 1 - Structural characterization of the universally conserved, heptameric Y-complex core. The 575 kDa Y-complex is the pivotal NPC architectural scaffold unit. Building on recently published work on fragments of the Y-complex it is now feasible to structurally characterize the entire complex. A combined approach using crystallographic and electron microscopic methods will be employed.
Aim 2 - Structural characterization of the species-specific Y-complex extensions. The human Y-complex contains three additional proteins, whose functional roles include NPC architecture and assembly. These proteins will be studied structurally in complex with the core components of the Y-complex.
Aim 3 - Structural characterization of the Nic96 complex. The Nic96 complex is only partially characterized, but it shares common ancestry with the Y-complex. It is widely accepted that it is pivotal to elucidate the Nic96 complex structure as it is, beside the Y-complex, the second large building block of the NPC scaffold structure.
Aim 4 - Assembly structure of the NPC and interactome. NPC subcomplexes have to interact to form the higher order assembly of the NPC, yet information on these inter-subcomplex contacts is still incomplete. To solve this problem, a yeast-two-hybrid based interactome will be generated sampling binary interactions between all nucleoporins.

Public Health Relevance

This study will lead to a structural understanding of the massive nuclear pore complex (NPC), the sole transport channel to and from the cell's nucleus and the passage route for many pathogenic virus, including HIV. This knowledge will open mid-term possibilities to influence transport properties, possibly to block viral entry to the nucleus. The NPC is also involved in a myriad of other human diseases, including cancer and cardiomyopathies, thus a molecular understanding of these mechanisms may provide new long-term therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM077537-06A1
Application #
8371894
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Flicker, Paula F
Project Start
2007-02-01
Project End
2016-07-31
Budget Start
2012-09-01
Budget End
2013-07-31
Support Year
6
Fiscal Year
2012
Total Cost
$386,525
Indirect Cost
$128,916
Name
Massachusetts Institute of Technology
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Ulrich, Alexander; Partridge, James R; Schwartz, Thomas U (2014) The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution. Mol Biol Cell 25:1484-92
Schwartz, Thomas (2013) Functional insights from studies on the structure of the nuclear pore and coat protein complexes. Cold Spring Harb Perspect Biol 5:
Bilokapic, Silvija; Schwartz, Thomas U (2013) Structural and functional studies of the 252 kDa nucleoporin ELYS reveal distinct roles for its three tethered domains. Structure 21:572-80
Andersen, Kasper R; Leksa, Nina C; Schwartz, Thomas U (2013) Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification. Proteins 81:1857-61
Bilokapic, Silvija; Schwartz, Thomas U (2012) 3D ultrastructure of the nuclear pore complex. Curr Opin Cell Biol 24:86-91
Brohawn, Stephen G; Partridge, James R; Whittle, James R R et al. (2009) The nuclear pore complex has entered the atomic age. Structure 17:1156-68
Whittle, James R R; Schwartz, Thomas U (2009) Architectural nucleoporins Nup157/170 and Nup133 are structurally related and descend from a second ancestral element. J Biol Chem 284:28442-52
Partridge, James R; Schwartz, Thomas U (2009) Crystallographic and biochemical analysis of the Ran-binding zinc finger domain. J Mol Biol 391:375-89
Leksa, Nina C; Brohawn, Stephen G; Schwartz, Thomas U (2009) The structure of the scaffold nucleoporin Nup120 reveals a new and unexpected domain architecture. Structure 17:1082-91
Boehmer, Thomas; Jeudy, Sandra; Berke, Ian C et al. (2008) Structural and functional studies of Nup107/Nup133 interaction and its implications for the architecture of the nuclear pore complex. Mol Cell 30:721-31

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