The overall goal of this proposal is to investigate the mechanism of NTF2-mediated Ran import. The Ran GTPase is a central component of an essential process in all eukaryotic cells -- nuclear transport. Results from this laboratory and others demonstrate that nuclear import of the small GTPase Ran is mediated by Nuclear Transport Factor 2 (NTF2), defining a novel Ran import pathway. Here, we will test three hypotheses based on our model for Ran import. Using in vitro biochemistry and a permeabilized cell assay, we will investigate the effect on Ran import of NTF2 sequestration and targeting of RanGDP to the nuclear pores. Additionally, we will test the prediction that RCC1 exchange activity is required for release of Ran from nuclear pores using quantitative exchange assays and a novel permeabilized cell assay. Finally, we will investigate the function in Ran transport of Nup 153, a candidate NTF2:RanGDP nuclear pore docking protein that is proposed to function in other transport pathways. Investigation of these hypotheses is likely to reveal new information as to the molecular interactions required for NTF2-mediated Ran transport, both in the soluble phase of transport and in the stationary phase at the nuclear pore. These new data may provide information critical to our further understanding of nuclear transport, a dynamic process absolutely required for life in all eukaryotic cells.
