The goal of this proposal is to elucidate the mechanism of Ran- dependent nuclear protein import. Several models for nuclear transport have been proposed that are mutually incompatible. A new model is suggested here that provides a solid basis to test the current assumptions of this import process. In particular, the role of the Ran:GDP/Ran:GTP gradient and the energy requirement for import will be addressed by the development of a new in vitro assay. By merging in vitro import assays of permeabilized cells with nuclear microinjection, the concentration of factors on both sides of the nuclear pore complex (NPC) can be manipulated. The role of an external binding site for Ran:GDP on the NPC will also be explored, by studying the role of NTF2 in docking and import. NTF2 is a factor necessary for nuclear protein import. It will be asked whether addition of exogenous NTF2 causes increased Ran:GDP docking to NPCs in permeabilized cells to test the assumption that NTF2 binding to Ran:GDP catalyzes translocation of the import complex. In vitro assays will be performed to determine whether NTF2 moves into the nucleus with Ran during import. Finally, the role of RanBP2 will be addressed. RanBP2 may constitute the Ran:GTP binding site at the NPC where Ran:GTP is hydrolyzed to Ran:GDP to allow import complex formation. This will be tested by blocking the Ran binding sites at RanBP2 with G19V Ran, a mutant unable to hydrolyze GTP, the measuring import rates. In addition, since RanBP2 may constitute the site of both Ran:GDP and Ran:GTP binding at the NPC, it will be determined whether Ran:GDP and Ran:GTP (G19V) are able to bind to the same pores by immunoelectron microscopy. As a result of these experiments, a more accurate picture of nuclear protein import can be obtained to clarify the mechanism of this process. HIV and other clinically relevant viruses manipulate nuclear transport processes to facilitate their replication. Understanding of the molecular mechanisms of nuclear transport may, therefore, identify novel targets for antiviral therapies.
