Using peptide arrays to define nuclear carrier binding sites on nucleoporins.

In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-beta. Crystallization studies by others have indicated that nuclear carriers such as importin-beta bind to phenylalanine-glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-beta identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-beta. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-beta binding to those repeats. In addition to these expected binding sequences for importin-beta, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-beta, indicating that additional Nup sequences may form binding sites for importin-beta.