The long term goal of this application is to understand the mechanisms by which cells localize nuclear proteins via a specific transport pathway. The proper regulation of nuclear protein transport is essential to maintenance of a normal cellular phenotype, developmental processes and the cellular response to the external environment. In this application, the initial steps in the transport of a nuclear protein from the cytoplasm to the nucleus will be investigated. The first identifiable step in nuclear protein import is docking of the nuclear protein at the cytoplasmic side of the nuclear pore complex. Cytoplasmic assembly of a receptor complex composed of two transport factors, p97 and the NLS receptor is believed to precede docking of the complex at the nuclear pore. Interaction of the receptor complex with the nuclear pore is mediated by binding of p97 to a nuclear pore complex protein. Subsequent release of the docked receptor complex and translocation to the nuclear interior requires the small GTPase Ran/TC4.
The specific aims of this application are designed to understand the molecular interactions between proteins of the receptor complex during the docking step of transport. The first two specific aims of this application are designed to characterize the physical structure of p97 to understand how it interacts with other components and directs docking.
These aims will be achieved by analysis of and epitope on p97 recognized by a specific monoclonal antibody to p97 that inhibits the translocation of receptor complexes into the nucleus. This epitope is believed to be partially determined by a post- translational modification of p97 in a domain that is required to remove the docked complex from its nucleoporin binding site. Expression of fragments of p97 in bacteria and mammalian cells will be used to identify the epitope domain on p97. The specific domain identified will be used as an affinity probe to identify transport factors that are involved in release of the docked complex from the pore. p97 is also a zinc binding protein. The role of zinc in establishing the structure and function of p97 will be analyzed by determination of the zinc binding domain. Analysis of zinc binding to deletion constructs of p97 will be carried out to identify the domain. Site directed mutagenesis will identify specific residues required for zinc binding. The zinc binding domain will be correlated with biochemical assays to identify binding domains for other transport factors on p97.
The third aim i s the characterization of a novel nuclear transport factor, p110, that was identified by interaction with p97. Preliminary characterization of the binding of p110 to p97 suggests that this protein may function by regulating the interaction of p97 with the nuclear pore and Ran-GTP. A cDNA clone for p110 will be isolated and sequenced. The role of p110 in protein import will be assayed in a permeabilized cell assay. Specific antibodies to p110 will be prepared to localize the protein in cells and study the interactions of p110 in cell lysates.
The fourth aim i s to develop a biochemical assay for assembly of the docking complex on isolated components of the nuclear pore. The temporal order of assembly of the docking factors is unknown as is the precise composition of the docking complex. These experiments will further our understanding at the molecular level of early events in the transport of proteins in the nucleus.
