Deciphering rules for nuclear trafficking

Compartmentalization of eukaryotic cells by the nuclear envelope into cytoplasm and nucleus necessitates efficient and selective passage of macromolecules through the nuclear pore complex. In human cells, 19 different Karyopherinβ (Kapβ, also known as Importins or Exportins) proteins transport macromolecules in and out of the nucleus by recognizing distinct nuclear localization or export signals (NLSs or NESs). Thus, Kapβs are critically involved in cellular processes such as gene expression, signal transduction, immune response, oncogenesis and viral propagation, all of which require proper nucleocytoplasmic targeting.

Research in our lab is directed towards understanding the physical and cellular mechanisms of Kapβs. Our long-term goals are to understand how the macromolecular nuclear traffic patterns coordinated by the 19 human Kapβs contribute to overall cellular organization. Why does the cell need so many different Kapβ? What are the signals that are recognized by each of the 19 Kapβs? Are cargoes of individual Kapβs random except for a common signal? Our research aims to answer these fundamental questions.

Only two classes of NLS are currently known. The lysine-rich classical-NLS was discovered in the 1980s and is recognized by the Kapα/Kapβ1 (also known as Importinα/Importinβ). In 2006, our lab defined a new NLS class termed the PY-NLS. Using structural, biochemical and bioinformatics approaches, we defined a set of physical rules for PY-NLS recognition by Kapβ2 (also known as Transportin) and used these to discover new candidate cargoes.

The only known NES is the leucine-rich NES, which is recognized by Kapβ CRM1. We have recently solved the crystal structure of CRM1 bound to leucine-rich NES containing cargo Snurportin 1. The structure explained the recognition of this general export signal, the more complex bipartite recognition of Snurportin 1 by CRM1 and the inhibition of CRM1 by Leptomycin B. The structure also revealed mechanisms of the different steps of nuclear export such as export complex assembly in the nucleus and disassembly in the cytoplasm.

With the exception of Kapβ1, Kapβ2 and CRM1, the other 16 Kapβs are currently known to each recognize only a few diverse sequences, making it extremely difficult to identify their NLSs/NESs. Nevertheless, large sizes, low sequence identities of Kapβs and identification of multiple substrate binding sites for several members all suggest that dozens of NLSs and NESs have yet to be discovered. Our Kapβ2 and CRM1 work now serve as model systems for future discovery of NLSs and NESs. We have discovered and are studying complex signals using a collection of physical rules rather than specific sequence motifs alone. This concept could be expanded to study numerous obscure targeting signals in eukaryotic cells and other biological recognition processes that involve linear recognition motifs with weak and obscure consensus sequences.