Dissection of a nuclear localization signal

The regulated process of protein import into the nucleus of a eukaryotic ce ll is mediated by specific nuclear localization signals (NLSs) that are rec ognized by protein import receptors. This study seeks to decipher the energ etic details of NLS recognition by the receptor importin alpha through quan titative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning a pproach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contribu tions of the second basic cluster in a bipartite NLS (similar to3 kcal/mol) as well as the energy of inhibition of the importin cu importin beta -bind ing domain (similar to3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha -importin beta complex. On thi s scale, a functional NLS has a binding constant of similar to 10 nra, wher eas a nonfunctional NLS has a 100-fold weaker affinity of 1 muM. Further co rrelation between the current in vitro data and in vivo function will provi de the foundation for a comprehensive quantitative model of protein import.