The investigator has developed an in vitro system which functions in authentic nuclear import. With this approach, the following results have already been established: 1, nuclear import is ATP-dependent and requires an intact nuclear envelope; 2, import is inhibited by wheat germ agglutinin binding to nuclear pores (implicating nuclear pore glycoproteins as possible participants in nucleocytoplasmic transport); and 3, nuclear import can be separated into two sequential steps, binding to the nuclear envelope and translocation through the pore. Binding is mediated by the nuclear targeting signal on the translocatable protein, and is independent of ATP and is not inhibited by wheat germ agglutinin. Translocation, however, requires ATP and is inhibitable by the lectin. During the tenure of this grant, the investigator will identify and chemically isolate the receptor which mediates specific binding of translocatable proteins, using chemical cross-linking techniques. Antisera will then be prepared against the receptor, and used for immunolocalization and functional studies. Also, the potential for receptor heterogeneity will be explored, by competition studies using synthetic nuclear targetting signal peptides. The communication between nucleus and cytoplasm is a critical feature of cellular life. On the one hand, it is imperative that the nuclear content of the cell remain segregated from the general cytoplasm, in order to permit the highly-ordered regulation of gene activity that is typical of eukaryotic cells; on the other hand, information, enzymatic machinery, and structural components must be provided to the nucleus from the cytoplasm. The results of this project will greatly increase our understanding of how this communication occurs and is regulated. Such understanding is a necessary prerequisite to experimental manipulations of cytoplasm-to-nucleus communication for specific biotechnical applications.
