The nuclear pore complex (NPC) is a massive macromolecular gateway, composed of proteins called nucleoporins (Nups), which makes bidirectional transport across the nuclear membrane possible. The core of the NPC can be readily described as being organized into concentric layers. The components of these layers do not exist independently of one another and the components of the NPC are assigned to each layer based upon function. The outermost layer consists of pore membrane proteins (POMs) that anchor the NPC to the nuclear membrane. The "coat" Nups form the next innermost layer and connect the POMs to the "adaptor" layer. A key player within the adaptor layer is Nup93, which forms a central hub by interacting with the other proteins of the adaptor layer as well as the nucleoporins of the innermost layer called the "transport channel". Four nucleoporins, all of which are essential for cell viability and nuclear transport, comprise the transport channel: Nup54, Nup58/45 (Nup45 is a splice variant of Nup58), and Nup62. Based on previous work in the Blobel laboratory, Nup58 is proposed to form the central "midplane" ring of the transport channel through which cellular traffic passes. The diameter of the midplane ring is predicted to double in diameter upon binding of Nup54 to Nup58. Additionally, Nup54 links Nup58 and Nup62 together within the transport channel and contains independent binding sites for both. Although the structures of individual nups and transport factors are well characterized, how different ring layers within the NPC interact and how the diameter of the midplane ring is modulated is poorly understood. The goal of this proposal is to determine the basis for interaction between the adaptor layer and the transport channel and how their interaction might modulate the opening and closing of the midplane ring.
Aim 1 : To determine the crystal structure of a complex of Nup93 with the triple complex of Nup54, Nup58, and Nup62. I will screen for crystals of a complex of Nup93 with Nup54, Nup58, and Nup62 using standard crystallization techniques and can already isolate a complex of these four nups using size-exclusion chromatography. Solving the structure of this complex will reveal how the adaptor layer interacts with the transport layer of the NPC and how Nup93 and the adaptor layer regulate the opening and closing of the Nup58 ring.
Aim 2 : To functionally characterize the interactions between the adaptor layer and the transport channel to determine how the adaptor layer may modulate ring opening and ring closing. I will use semi-permeabilized cell nuclear import assays with gold particles conjugated to nuclear localization sequence-GFP fusion proteins to investigate whether or not mutants that disrupt the interaction between Nup93 and Nup54, Nup58, and Nup62 affect nuclear import. Additionally, I will characterize the effect of the mutations on the interaction of Nup93 with the transport channel using size-exclusion chromatography and isothermal titration calorimetry. Finally, using western blotting and mass spectrometry, I will probe for additional interactions between the adaptor layer and transport channel.

Public Health Relevance

The nuclear pore complex (NPC) is a gateway that is conserved from yeast to man and regulates the flow of cellular traffic into and out of the nucleus. Malfunctions of the nuclear pore complex have been implicated in many human diseases including viral infection, primary biliary cirrhosis, infantile bilateral striatal necrosis, tripleA syndrome, and cancer (Cronshaw and Matunis, 2004). The goal of this proposal is to reveal interactions within the central transport channel of the NPC and to understand the function of the NPC by determining how it opens and closes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM105207-01
Application #
8456423
Study Section
Special Emphasis Panel (ZRG1-F04-D (20))
Program Officer
Flicker, Paula F
Project Start
2013-02-01
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
1
Fiscal Year
2013
Total Cost
$52,190
Indirect Cost
Name
Rockefeller University
Department
Anatomy/Cell Biology
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Wang, Jimin; Wing, Richard A (2014) Diamonds in the rough: a strong case for the inclusion of weak-intensity X-ray diffraction data. Acta Crystallogr D Biol Crystallogr 70:1491-7