Domains of Importin-α2 required for ring canal assembly during Drosophila oogenesis

Null-mutation in Drosophila importin-α2, such as the deficiency imp-α2D14, causes recessive female sterility with the formation of dumpless eggs. In imp-α2D14 the transfer of nurse cell components to the oocyte is interrupted and the Kelch protein, an oligomeric ring canal actin organizer, is normally produced but fails to associate with the ring canals resulting in their occlusion. To define domains regulating Kelch deposition on ring canals we performed site-directed mutagenesis on protein binding domains and putative phosphorylation sites of Imp-α2. Phenotypic analysis of the mutant transgenes in imp-α2D14 revealed that mutations affecting the Imp-β binding-domain, the dimerization domain, and specific serine residues of putative phosphorylation sites led to a normal or nearly normal oogenesis but arrested early embryonic development, whereas mutations in the nuclear localization signal (NLS) and CAS/exportin binding domains resulted in ring canal occlusion and a drastic nuclear accumulation of the mutant proteins. Deletion of the Imp-β binding domain also gave rise to a nuclear localization of the mutant protein, which partially retained its function in ring canal assembly. Thus, we propose that mutations in NLS and CAS binding domains affect the deposition of Kelch onto the ring canals and prevent the association of Imp-α2 with a negative regulator of Kelch function.