The nuclear pore complex (NPC) is an efficient and selective filter that mediates all nucleocytoplasmic transport. The basic structure of the NPC is a cylindrical tube lined with proteins rich in hydrophilic natively disordered regions containing degenerate repeated phenylalanine-glycine (FG) motifs termed """"""""FG nups"""""""". It is these FG nups that form the """"""""business end"""""""" of the NPC, allowing the passage of transport factors and their cargo while inhibiting the passage of other macromolecules. Despite significant recent advances, there are many outstanding questions and controversies about the behavior of the FG nups within the NPC. In particular, which features of the FG nups are important for their role in selective transport? We have developed an in vitro model system which mimics key features of the NPC, namely that FG nups line the surface of the central tube, a structure that is ~ 35x50 nm. We have covalently attached the FG repeat portion of two archetypal FG nups to 40 nm radius latex beads in an orientation and density comparable to that found in the NPC. We will first use cryo-electron microscopy to discern the structure that FG nups form on the surface of the beads and monitor changes in their structure upon binding of transport factors and other proteins. We will then employ light microscopy to monitor changes in the dynamics of FG nups and their interactions with transport factors concurrent with the observed structural changes. These experiments will give insight into an important class of natively disordered proteins, and will help in the rational design of NPC based artificial filters for protein purification. The nuclear pore complex (NPC)is the gate that regulates which macromolecules can enter and leave the nucleus. A better understanding of how the NPC is able to allow some macromolecules to pass through the NPC while blocking other macromolecules could help in the treatment of related diseases and lead to the rational design of NPC based filters that would be used in the production of biomedically important proteins. We will use two different kinds of micrsocpy to visualize the structure of a key portion of the NPC and determine which structural features are important for its function.
| Hough, Loren E; Dutta, Kaushik; Sparks, Samuel et al. (2015) The molecular mechanism of nuclear transport revealed by atomic-scale measurements. Elife 4: |
| Tetenbaum-Novatt, Jaclyn; Hough, Loren E; Mironska, Roxana et al. (2012) Nucleocytoplasmic transport: a role for nonspecific competition in karyopherin-nucleoporin interactions. Mol Cell Proteomics 11:31-46 |
