Identification of specific binding proteins for a nuclear location sequence

Abstract

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm1. Nuclear proteins larger than relative molecular mass 20,000–40,000 are probably actively transported across the envelope through the nuclear pore complex2,3 and are directed by specific nuclear location sequences (NLS) in the proteins4. NLS mediate the nuclear import of isolated nuclear proteins after microinjection into whole cells5,6 and the nuclear accumulation of chimaeric proteins7–9 or of non-nuclear proteins conjugated to synthetic peptides10–12. The best-characterized NLS is the simian virus 40 large T-antigen sequence4,13,14. We have identified two proteins of rat liver by chemical cross-linking that interact with a synthetic peptide containing this sequence: this interaction is specific for a functional NLS, is saturatable, and high affinity. The binding proteins are present in a post-mitochondrial supernatant, in nuclei and in a nuclear envelope fraction, which is consistent with a role in the transport of nuclear proteins from the cytoplasm to the nucleus.