Abstract
Small molecules - less than 40 kD - diffuse freely through nuclear pores between the cytoplasm and nucleus. Large proteins and macromolecular complexes require active transport across the nuclear envelope via soluble transport factors or carrier molecules. Some carrier molecules themselves form complexes as they cycle between the nucleus and cytoplasm. One such transport factor is NTF2, which is known to play a primary role in the maintenance of the Ran gradient, but the oligomeric state of NTF2 and its function in transport are not well established. We observe that NTF2 exists as a monomer-dimer equilibrium in the cell, and we characterize several NTF2 mutants to investigate the influence of NTF2 dimerization on nucleocytoplasmic transport. We apply two-photon activation in vivo to examine the transport of photoactivable GFP-tagged carrier proteins. We also use fluorescence fluctuation spectroscopy and brightness analysis in the cell to investigate the oligomerization of the NTF2 mutants. We establish the efficacy of brightness analysis in a cell free expression system and use it to check oligomerization of NTF2 and its mutants in vitro. This work is supported by NIH grant R01GM064589.
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