Transport of RNAs and proteins and between the nucleus and cytoplasm occurs through large proteinaceous structures that are embedded in the nuclear envelope, called nuclear pore complexes (NPCs). These 125 MDa complexes restrict free diffusion of molecules or particles with a diameter larger than 9 nm, corresponding to the size of a globular protein of 40-60 kD. However, complexes of up to 25 nm in diameter (several megadaltons) are efficiently transported provided they carry a nuclear import or export signal. 

Nucleocytoplasmic transport is conducted by distinct classes of soluble transport receptors that interact with both cargo and nuclear pore complex. One class consists of importins and exportins, a superfamily of transport receptors that are responsible for the majority of nuclear transport pathways identified to date. Importins mediate import of several different classes of (ribonucleo) proteins, while exportins mediate nuclear exit of proteins, tRNAs and U snRNAs. 

Importin and exportin mediated transport is dependent on a signal within the transport substrate. Examples of import signals are the basic nuclear localization signal (NLS) that is found in many nuclear proteins. Similarly, the nuclear export signal (NES) mediates rapid nuclear export. Leucine-rich-type NESs have recently been identified in a diverse range of cellular proteins that include cell cycle regulators, stress response signal transducers and mediators of apoptosis. There are at least nine different importins and five exportins, each recognizing a different or a different subset of transport signals. 
 

In the lab Gerard Grosveld (St. Jude's Children's Research Hospital, Memphis, USA), we identified proteins that interacted with the nucleoporin CAN/Nup214. One of them was the human homologue of a yeast protein named CRM1, that we proposed to be a new transport factor. This proposal was based on experimental data, and on sequence homology with the import receptor importin b. We identified a group of related proteins with homology to both CRM1 and importin b that was proposed new family of transport factors (Fornerod et al., 1997a).

In the lab of Iain Mattaj (EMBL, Heidelberg, Germany), together with Mutsuhito Ohno, we established that CRM1 was an export receptor for "leucine rich" nuclear export signals (see above). We showed that CRM1 binds directly to the export signal, and that this can be inhibited by the fungal cytotoxin leptomycin B (LMB). Leptomycin B was also shown to directly bind to CRM1. NES binding was only stable upon cooperative binding of the small GTPase Ran in its GTP-bound form. This suggested the mechanism with which the RanGTP gradient across the nuclear envelope directs transport of NES-containing (ribonucleo) proteins, and we proposed that this mechanism would be more generally applicable to nuclear export processes (Fornerod et al., 1997b).

With Gert-Jan Arts, post-doc in Iain Mattaj's lab, we identified the nuclear export receptor for tRNAs, exportin-t, another member of the importin b family. Similar to CRM1, exportin-t bound directly to its tRNA cargo in a RanGTP-dependent manner (Arts et al., 1998). 

Refocussing on the nuclear pore complex, we have extended the analysis of the CAN/CRM1 interaction, and demonstrated that the interaction dependents on RanGTP, suggesting a role of this interaction in export complex disassembly and CRM1 reimport (Askjaer et al., 1999).

It has recently become clear that many cellular processes, such as signal transduction, cell cycle progression and apoptosis are regulated on the level of nuclear transport. Much of this progress was based on the identification of transport signals, transport receptors, and understanding of mechanisms of the directionality of transport. 

Such basic understanding is clearly lacking of the role of the NPC in nucleocytoplasmic transport. Therefore we study the basic function of nuclear pore complex in nucleocytoplasmic transport pathways. In parallel, we are exploring to what extend the NPC functions as a regulator of transport and as a regulator of other cellular processes.

We are using a combination of biochemistry and cell biology to understand the mechanism of nucleocytoplasmic transport and NPC function. Biochemical methods can define transport interactions on a molecular level, while functional cell biological assays are necessary to address the role of the nuclear transport interactions.

One of the experimental systems used is nuclear reconstitution. Egg extracts of the african clawed frog, Xenopus laevis, support efficient nuclear formation around demembrenated sperm chromatin in vitro. These nuclei contain numerous nuclear pore complexes, and actively import protein substrates. Also the DNA can undergo one round of replication. 

Nuclear transport can be monitored by accumulation of exogenously added fluorescently labelled substrate (see Figure below). Mutant NPCs can be created by removing NPC components from the egg extract prior to nuclear assembly. Those mutant NPCs can be studied for transport and other defects. Add back of purified or recombinant nucleoporins can demonstrate specificity, and is used to identify regions in the protein responsible for the observed effects.

Maarten Fornerod and Mustuhito Ohno (2002). Exportin-mediated nuclear export of proteins and ribonuleoproteins. Res. Probl. Cell Diff. 35:67-91 (in press).

Tobias C. Walther, Maarten Fornerod, Helen Pickersgill, Martin Goldberg, Terry D. Allen and Iain W. Mattaj (2001). The Nucleoporin Nup153 is Requred for Nuclear Pore Basket Formation, Nuclear pore Complex Anchoring and Import of a Subset of Nuclear Proteins. EMBO J. 20:5703-5714.

Ludwig Englmeier, Maarten Fornerod, F. Ralf Bischoff, Carlo Petosa, Iain W. Mattaj and Ulrike Kutay (2001). RanBP3 facilitates nuclear protein export by stabilising the interaction between certain export substrates and CRM1. EMBO Rep. 2:926-932.

Jörg Hamm and Maarten Fornerod (2000). Anti-idiotype RNAs that mimic the leucine-rich nuclear export signal and specifically bind to CRM1/exportin 1. Chem. Biol. 7:345-54.

Peter Askjaer, Angela Bachi, Matthias Wilm, Ralf Bischoff, Daniel Weeks, Vera Ogniewski, Mutsuhito Ohno, Christof Niehrs, Jorgen Kjems, Iain W. Mattaj and Maarten Fornerod (1999). RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase. Mol. Cell. Biol. 19: 6276-6285.

Colin Pritchard, Fornerod M., Lawryn L. Kasper, and Jan M. van Deursen. (1999). RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains. J. Cell Biol. 145:237-54.

Gert-Jan Arts, Maarten Fornerod and Iain W. Mattaj (1998). Identification of a nuclear export receptor for tRNA. Curr. Biol. 8:305-14.

Mutsohito Ohno, Maarten Fornerod and Iain W. Mattaj (1998). Nucleocytoplasmic transport: the last 200 nanometers. Cell 92:327-36.

Maarten Fornerod, Mutsohito Ohno, Mironu Yoshida and Iain W. Mattaj (1997b). CRM1 is a nuclear export receptor for leucine-rich nuclear export signals. Cell 90: 1051-1060.

Maarten Fornerod, Jan van Deursen, Sjozèf van Baal, Donna Davis, K. Gopal Murti and Gerard Grosveld (1997a). The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88. EMBO J. 16: 807-816.