Molecular Basis of Ran-Mediated Nuclear Transport

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DESCRIPTION (provided by applicant): The absolute compartmentalization of the genetic material within the nucleus of the eukaryotic cell creates a critical control point for intracellular signaling. Whenever expression of specific genes is regulated in response to an intra- or extraceullar signals, information is transferred across the nuclear envelope. In many cases this information is transmitted as specific proteins that enter the nucleus to elicit changes in gene expression. Despite the biological importance of this process, the mechanism of this signal dependent nuclear targeting and translocation is not understood at the molecular level. In order for these events to serve as therapeutic targets the detailed molecular mechanism must be delineated. The broad long-term objective of this proposal is to understand how soluble transport factors cooperate with the nuclear pore complex to mediate bidirectional nuclear transport. This study combines in vivo analyses of the highly conserved transport factors in the budding yeast, S. cerevisiae, with quantitative analysis of protein-protein interactions and microinjection into Xenopus oocytes to learn how cargoes are targeted to and delivered into the nucleus. The specific aims of this proposal are to: 1) Analyze molecular interactions between NTF2 and the nuclear pores that are required to translocate NTF2 through pores and exploit this analysis to distinguish between current models for transport through the nuclear pore complex; 2) Examine the mechanism of NLS cargo delivery into the nucleus; and 3) Investigate how phosphorylation within NLS sequences modulates protein import. Results from these experiments will provide novel insights into the mechanism of nucleocytoplasmic transport. The health-relatedness of this proposal is two-fold. First, activated signal transduction pathways send signals to the nucleus in order to respond to stimuli and activate transcription. This transport step may represent an unexploited target for blocking specific cellular signals as well as the unregulated signals that arise in transformed cells. Second, viruses that infect human cells exploit the endogenous nuclear transport machinery to gain entry to the nucleus. A more detailed understanding of the machinery that mediates nuclear transport may provide novel targets for anti-viral therapies.