Analysis of nuclear organization: Nucleocytoplasmic transport and chromatin remodeling

The nuclear pore complex (NPC) mediates the transport of macromolecules between the nucleus and cytoplasm. To better understand the mechanism of translocation through the NPC, I analyzed interactions between transport receptors and the NPC proteins, as well as the structural organization of the NPC. To this end, I developed a method that allows the in situ detection of protein interactions in the budding yeast Saccharomyces cerevisiae. In this microscopy-based assay, protein interactions are probed by fluorescence resonance energy transfer (FRET) between the enhanced cyan and yellow fluorescent proteins (ECFP, EYFP) fused to the target proteins.; Translocation pathways were investigated by assessing the patterns of interactions between transport receptors (importins and exportins) and proteins of the NPC (nucleoporins, nups). A panel of yeast strains each expressing functional receptor-ECFP and nup-EYFP fusions was generated and analyzed for FRET. The importin Pse1/Kap121 and the exportin Msn5 were found to have some common and some specific points of contact in the NPC. Kap121-Nup interactions were also examined by a series of immunoprecipitation experiments. The results indicate that an importin and exportin have overlapping but distinct translocation pathways.; To study the structural organization of the NPC, FRET was measured between the fluorescent proteins expressed as fusions to distinct nucleoporins. Thirteen FRET interactions were identified; independent genetics experiments were consistent with the results. Analysis of the spatial requirements of the FRET signals demonstrated the specificity of the assay. A refined molecular model of the NPC is proposed based on the integration of the FRET results with existing data.; In a separate project, the function of the yeast chromatin-remodeling complex RSC was investigated. Rsc9 was identified as a novel RSC component and was further characterized. Genome-wide localization analysis indicated that the Rsc9 targets include genes regulated by stress; two stress treatments resulted in genome-wide changes in Rsc9 occupancy. Northern analysis further demonstrated that Rsc9 is involved in both the repression and activation of genes regulated by the TOR signaling pathway. The results illustrate the response of a chromatin-remodeling factor to signaling cascades and have implications for the mechanism of RSC function.