Characterization of Nuclear Localization Signals of Karyopherin-Mediated Nuclear Import

Nucleocytoplasmic transport is mediated by Karyopherin beta (Kap beta) proteins in a Ran-dependent manner. Ten import Kap betas recognize their cargos through the nuclear localization signals (NLSs) and carry them into the nucleus. Recent structural and biochemical work on Kap beta2 (or Transportin) and its well-characterized hnRNP A1-NLS (or M9NLS) reveal that NLSs recognized by Kap beta2 are structurally disordered, have overall positive charges and contain a loose N-terminal hydrophobic or basic motif followed by a C-terminal conserved R/H/KX₍₂₋₅₎PY motif. The newly defined PY-NLSs are further divided into two subclasses: hydrophobic or basic PY-NLSs (hPY or bPY). Bioinformatic searches using these physical characteristics predicted 81 new PY-NLSs. Of the 77 tested new PY-NLSs, 13 showed strong binding to Kap beta2, 8 showed moderate binding and 56 have very weak or no binding. Comparison of Kap beta2 in complex with hnRNP A1 and M NLSs suggest that PY-NLSs are multivalent and each epitope has different contribution to the overall binding energy, which lead to the design of the chimeric M9M peptide. M9M as a Kap beta2-specific inhibitor mislocalizes the Kap beta2 cargos, hnRNP A1, HuR and hnRNP M but has no effect on HDAC1, a cargo for Impα/β pathway. Unexpected redundant import pathways for NXF1 are also discovered using M9M peptide. The N-terminal disordered region of human NXF1 contains NLSs for Imp beta, Kap beta2, Imp4, Imp11 and Imp alpha. Mutation of the NLSs in NXF1 abolished binding to the Karyopherins, mislocalized NXF1 to the cytoplasm and significantly compromised its mRNA export function. Sequence examination of NXF1 from divergent eukaryotes and the interactions of NXF1 homologs with various Karyopherins have revealed the redundancy of nuclear import pathways for NXF1 increased progressively from fungi to nematodes and insects to chordates.