Purification of protein A-tagged yeast Ran reveals association with a novel karyopherin beta family member, Pdr6p

The small GTPase Ran (encoded by GSP1 and GSP2 in yeast) plays a central ro le in nucleocytoplasmic transport. GSP1 and GSP2 were tagged with protein A and functionally expressed in a gsp1 null mutant. After affinity purificat ion of protein A-tagged Gsp1p or Gsp2p by IgG-Sepharose chromatography, kno wn karyopherin beta transport receptors (e.g. Kap121p and Kap123p) and a no vel member of this protein family, Pdr6p, were found to be associated with yeast Ran. Subsequent tagging of Pdr6p with green fluorescent protein revea led association with the nuclear pore complexes in vivo, Thus, functional t agging of yeast Ran allowed the study of its in vivo distribution and inter action with known and novel Ran-binding proteins.