Nuclear pore complexes (NPCs), made up of nucleoporins, are responsible for the exchange of macromolecules between the nucleus and cytoplasm. The NPC plays both a structural and
regulatory role, influencing gene expression and other nuclear processes. Karyopherins are soluble transport factors that recognize specific protein sequences and import or export
designated molecules through the NPC. Given that major pathological cellular processes are associated with altered nucleocytoplasmic transport, and many viruses target components of
the nucleocytoplasmic transport pathway, nucleoporins and transport factors are key targets for drug therapy.
Michael Rout (pictured left, The Rockefeller University),
John Aitchison (Seattle Biomedical Research Institute),
Yuh Min Chook (University of Texas Southwestern), and
Andrej Sali (University of California, San Francisco), the Principal Investigators of the grant entitled “PSI:Biology Partnership
Nucleocytoplasmic Transport: A Target For Cellular Control” (NPCXtals), have chosen
targets for structure determination and functional studies from among the comprehensive inventories of the NPC molecular components and transport factors. Structural insights will aid
in the determination of the fundamental mechanisms of these two key regulatory areas of nucleocytoplasmic transport, specifically providing a map of nuclear basket structure-function
relationships and establishing karyopherins as targets for cellular control.
The structural efforts of NPCXtals researchers have been expanded through collaboration with the PSI:Biology High-Throughput Center for Structure Determination, New York Structural Genomics Research Consortium (NYSGRC). Building on previous work and experience, NYSGRC has expressed and purified components of the Nuclear Pore Complex (NPC), as well as import factors and their cargos. To increase protein yields for nucleoporin targets, NYSGRC identifies interacting domains for binding that would lead to complex formation and allow for subsequent co-expression and co-purification. This collaboration, which was initially established during PSI-2, has yielded a number of crystal structures, among them the NPC components Nup116 from Candida glabrata and the N-terminal portion of Nup192 from S. cerevisiae.
Additional research focuses on the nucleocytoplasmic transporters, karyopherins. The Chook, Rout, and Aitchison Labs have done extensive work on elucidating the structural and cellular mechanisms of this class of molecules. Karyopherins function by recognizing particular areas, nuclear localization sequences (NLS) and nuclear export sequences (NES), in their target molecules. To dissect the determinants of nuclear localization signal peptide recognition, the Chook Lab and NYSGRC are working with a structurally established karyopherin, Kapb2, expressed and purified in complex with a range of cargo peptides.
Four of the groups collaborating in this PSI:Biology Partnership are involved in the NIH-supported National Center for Dynamic Interactome Research (www.ncdir.org). Led by Dr. Rout, several laboratories, including the Chait, Sali and Aitchison Labs, are developing methodology to obtain a comprehensive map of dynamic interactions of macromolecular complexes in the cell. These tools will allow researchers to explore the utility of the technology for functional elucidation of complex biological processes, with an initial focus on the processes related to cell cycle control, transcription, oncogenesis, and viral infection. These partnerships have the opportunity to inform each other and create a valuable synergy to uncover pivotal relationships, both within the NPC and between the NPC and transport-related molecules and sequences.
More information about technology developed by the NPCXtals Partnership can be found in the Featured Technology article, “Chaperone-Enabled Structural Biology.”