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 complex^2,3 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 cells^5,6 and the nuclear accumulation of chimaeric proteins^7–9 or of non-nuclear proteins conjugated to synthetic peptides^10–12. The best-characterized NLS is the simian virus 40 large T-antigen sequence^4,13,14. 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.