Systems analysis of nuclear transport

The nucleus is a distinguishing feature of eukaryotic cells. Traffic is driven by the gradient of the small GTPase Ran, which exists in its GTP-bound form largely in the nucleus and GDP-bound form largely in the cytoplasm. Soluble transport receptors, called Karyopherins, act to bring cargo into the nucleus(importins) or back to the cytoplasm(exportins).; Using a combined experimental/in silico approach, we show that the adapter protein Impalpha acts as a major limiting reactant for nuclear transport. Ran and the transport receptor for Ran, NTF2, also act as limiting reactants to a lesser degree. Surprisingly, Impbeta and the guanine exchange factor RCC1 inhibit import at higher levels. Results from the computer model show that excess Impbeta lowers the RanGTP gradient by binding to RanGTP in the nucleus and carrying it back to cytoplasm without cargo, a process called futile cycling. We next used the model to generate a testable prediction based on this mechanism that was verified experimentally. The simulation also showed that the inhibitory effect of RCC1 was based on the sequestration of RanGTP. The model made a quantitative prediction that excess RCC1 should lead to accumulation of Ran in the nucleus, and this was also experimentally verified.; In the second phase of my project, I developed a more detailed model that included an explicit representation of the nuclear pore complex as well as the export pathway mediated by CRM1, the major exportin in mammalian cells. We used the model to look at steady-state cargo accumulation in nuclear transport as well as investigate the difference between direct and adapter-mediated transport. Computer simulation shows that, surprisingly, adapter mediated import provides no advantage in driving a nuclear cargo gradient. We verified this prediction using a variety of recombinant protein cargoes. Further work showed that adapter-mediated import may provide a regulatory advantage by increasing the dynamic range of control over import. Lastly, by extending our computer model to include a 3-compartment representation of the nuclear pore complex, we demonstrate how an increasing gradient of Impbeta-nucleoporin affinity, previously shown in vivo, can function to increase the efficiency of nuclear import.