The nuclear transport of proteins and RNAs occurs as a family of parallel and converging import and export pathways. We propose to identify and characterized novel biochemical intermediates that are responsible for U1 snRNP and calmodulin (CaM)-specific import pathways. using biochemical and molecular approaches. The purification and analysis of pathway- specific transport factors using in vitro assays will allow us to reconstruct the molecular mechanisms responsible for the targeting and translocation of these recently discovered pathways. Also, our kinetic analysis of import pathways will be extended to the bi-directional transport of import and export substrates. Nuclear transport is a fundamental cell process that plays a central role in the growth, regulation, and division of healthy, diseased, and infected tissues. The elucidation of molecular mechanisms pertaining to this process should result in rational approaches to disease treatment and prevention. We have, for example, been able to control HIV-1 infection in tissue culture cells by arresting the nuclear transport of the virion. 1. Characterization and in vitro reconstitution of U snRNP import. In Xenopus oocytes the import of U1-related U snRNPs proceeds by a kinetically distinct pathway. We now propose to reconstitute U1 snRNP import using an cell extract-dependent permeabilized tissue cell assay. We will fractionate cytosolic extracts for components that re required for efficient U1 snRNP import. We will also specifically investigate the role of hsc/hsp 70 in RNP import by depleting extracts of these proteins. We may be able to identify independent fractions that mediate only the targeting and those that mediate the subsequent translocation. Ultimately, we hope to biochemically purify transport factors and begin a molecular analysis of their structure and function. 2. Nuclear transport of calmodulin CaM). Preliminary results indicate that CaM import occurs by a novel mechanisms which may prove to be a general transport pathway for small regulatory proteins. We will use an in vitro Cam import assay to fractionate and purify cytosolic transport factors that are sufficient and necessary to reconstitute the in vivo mechanism of CaM import. Putative CaM transport factors will be analyzed by mass spectrophotometric fingerprinting and with specific polyclonal antibodies. We will also test our hypothesis that CaM shuttles in and out of nuclei by heterokaryon analysis. How the import of CaM differs from other pathways will be studied by direct competition in vivo and by reconstitution in vitro. 3. Kinetics of simultaneous import and export. The import of both P(Lys)-BSA and U1 snRNP are saturable, as is the export of tRNAs and ribosomes. It is not known how these distinct import and export pathways are coordinated through individuals pore complexes. We propose to perform a complete competition kinetic analysis of simultaneous bi-directional transport by double injections into oocytes. Preliminary results indicate that ribosome export and protein import pathways compete at the pore complex for limiting factors.
