Biochemical Analysis of the Protein-Protein Interactions Involved in Karyopherin-Mediated Transport Across the Nuclear Pore Complex

Nucleocytoplasmic transport occurs through the nuclear pore complex (NPC), which in yeast is a highly symmetric ~50 MDa complex consisting of approximately 30 different proteins. Small molecules can freely exchange through the NPC, but macromolecules larger than ~40 kDa such as proteins, mRNAs, and ribosomal subunits must be aided across by shuttle proteins (karyopherins, or Kaps). Kap-mediated transport involves FG-nups, a family of NPC proteins. While much has been learned about the mechanism of nucleocytoplasmic transport, many details are still unknown; perhaps among the most important missing details is the binding kinetics of almost all the transport relevant interactions, due to significant technical challenges. The aim of this work is to analyze the protein-protein interactions involved in Kap-mediated transport across the NPC, using biochemical, biophysical, and cell biological approaches. Yeast karyopherins, model cargoes, and full-length FG-nups are enriched from bacteria, and their affinities are studied quantitatively. The presence of competitor proteins and changes in bait protein distribution are seen to effect apparent affinity of these interactions. The relevance of the in vitro Kap/NLS-cargo binding measurements is confirmed with a nucleocytoplasmic import assay that allows quantitative measurements of import to be made within single living cells. Trends observed in vitro for Kap/FG-nup interactions were consistent with ex vivo observations of interactions of transport factors with Xenopus oocyte NPCs and also with in vitro measurements of transport through a synthetic NPC-based filter. This work has suggested a role for factors such as non-specific competition in determining the kinetics and selectivity of transport