The sequestering of genetic material in the nucleus by eukaryotic cells provides a powerful mechanism for the regulation of gene expression and other cellular processes through the selective translocation of proteins between the nucleus and the cytoplasm. Recently, the regulated transport of proteins across the nuclear envelope has been recognized as a crucial step in an increasing number of cellular processes. Elucidation of the mechanisms of regulated protein translocation through nuclear pores requires a detailed definition of the signals that mark a macromolecular complex for nuclear import or export. The best-characterized mechanism for translocation across the nuclear envelope is for protein import that depends on the 'classical' nuclear localization signal or NLS. This NLS consists of a cluster of basic residues (monopartite) or two clusters of basic residues separated by 10-12 residues (bipartite). This signal is recognized by the heterodimeric import receptor complex comprising importin a and importin b. We have recently developed a quantitative assay that measures the affinity of the import receptors for an NLS sequence at equilibrium in solution. With this fluorescence assay, we have begun to reconstitute the molecular reactions of protein import in vitro to provide a detailed thermodynamic description of the translocation reaction. A complete understanding of nuclear import signals requires a quantitative model for the import reaction that correlates NLS amino acid sequence, in vitro interaction energies, and in vivo functionality. A detailed description of the energetic requirements for an NLS sequence will facilitate the recognition of these sequences in protein primary structure as well as suggest possible modes for the regulation of protein import. In this proposal, we explore the mechanism of 'classical' nuclear import through quantitative analysis, correlation of in vitro measurements with in vivo function, and delineation of the specificity of the classical import pathway.
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