A role for the basic patch and the C terminus of RanGTP in regulating the dynamic interactions with importin beta, CRM1 and RanBP1.
Transport of macromolecules between the nucleus and cytoplasm involves the recognition of intrinsic localization signals by either import or export receptors. The interaction of the receptors with their cargo is regulated by the small GTPase Ran in its GTP bound state. We have investigated the interaction of RanGTP with the import factor, importin beta, the export factor, CRM1, and the Ran binding protein, RanBP1, in solution. Importin beta specifically protected residues in the switch regions and basic patch region of Ran against proteolytic cleavage, whereas RanBP1 protected the C terminus. Moreover, the binding of importin beta induced a conformational change in the structure of Ran leading to an exposure of the C terminus and stimulated the binding of RanBP1. Mutating the basic patch (HRKK(142)) of Ran resulted in an increased binding of RanBP1 and weakened importin beta binding. In contrast to wild-type Ran, the mutant Ran could be released from importin beta independently of importin alpha. These data provide experimental support for a model in which the accessibility of the C terminus of Ran is influenced by an intramolecular interaction between the basic patch and the C-terminal acidic DEDDDL(216) motif. Binding of importin beta probably disrupts this interaction causing an exposure of the C-terminal extension, which is favorable for RanBP1 binding. Interestingly, basic patch mutations abolish CRM1 interaction, indicating that the determinants for RanGTP binding to the export factor, CRM1, is different from the import factor, importin beta.
Nilsson
J
J
Department of Molecular and Structural Biology, University of Aarhus, AarhusC, DK-8000, Denmark.
Askjaer
P
P
Kjems
J
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D028884:Karyopherins; D046911:Macromolecular Substances; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D023302:Protein Footprinting; D018160:Receptors, Cytoplasmic and Nuclear; D020931:ran GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000097606:Exportin 1 Protein
D002352:Carrier Proteins; D028884:Karyopherins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein; C091775:ran-binding protein 1