Understanding complete transport cycles mediated by importin beta-type nuclear transport receptors in situ and in living cells

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Many molecular components of the nucleocytoplasmic transport machinery have been identified over the last ten years and we have a good mechanistic understanding of this process which even extends to the structural level at atomic resolution (Conti and Izaurralde, 2001). However, formal proof of the central hypothesis of the driving force of nucleocytoplasmic transport, the existence of a RanGTP gradient across the nuclear envelope (NE) is still lacking. Moreover, we have currently no quantitative understanding of the kinetic parameters of the transport process in the context of the intact cell and are just beginning to understand its capacity and the mechanism of translocation through the nuclear pore complex itself (Ribbeck and Görlich, 2001, Rabut and Ellenberg, 2001). This project is aimed to address theses central open questions of nucleocytoplasmic transport. It will use a combination of biochemical, cell biological, light microscopy and mathematical modeling expertise uniquely combined in the laboratories of the two applicants. By purifying and fluorescently labeling all the components of the importin beta (Impb) mediated transport cycle, we will measure all the kinetic parameters of this reaction separately both in vitro and in situ (using permeabilized and live cells, and state of the art confocal microscopy) and finally combine them in a comprehensive mathematical model of nucleocytoplasmic transport. This model should ulitmately explain all the experimentally observed kinetics and can be used to predict the behaviour of the transport machineery and so far unknown limiting parameters. In addition, this project will use fluorescence energy transfer probes to directly visualize the RanGTP gradient as well as the spatiotemporal distribution of key protein complexes if the Ran system.