A conformational change in PBX1A is necessary for its nuclear localization

The fly homeodomain (HD) protein EXTRADENTICLE (EXD) is dependent on a seco nd HD protein, HOMOTHORAX (HTH), for nuclear localization. We show here tha t in insect cells the mammalian homolog of EXD, PBX1A, shows a similar depe ndence on the HTH homologs MEIS1, 2, and 3 and the MEIS-like protein PREP1, Paradoxically, removal of residues N-terminal to the PBX1A HD abolishes in teractions with MEIS/PREP but allows nuclear accumulation of PBX1A. We use deletion mapping and fusion to green fluorescent protein to map two coopera tive nuclear localization signals (NLSs) in the PBX HD. The results of DNA- binding assays and pull-down experiments are consistent with a model whereb y the PBX N-terminus binds to the HD and masks the two NLSs, In support of the model, a mutation in the PBX HD that disrupts contact with the N-termin us leads to constitutive nuclear localization. The HD mutation also increas es sensitivity to protease digestion, consistent with a change in conformat ion. We propose that MEIS family proteins induce a conformational change in PBX that unmasks the NLS, leading to nuclear localization and increased DN A-binding activity, Consistent with this, PBX1 is nuclear only where Meis1 is expressed in the mouse limb bud. (C) 2000 Academic Press.