Domains of Importin-alpha2 required for ring canal assembly during Drosophila oogenesis.
Null-mutation in Drosophila importin-alpha2, such as the deficiency imp-alpha2(D14), causes recessive female sterility with the formation of dumpless eggs. In imp-alpha2(D14) 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-alpha2. Phenotypic analysis of the mutant transgenes in imp-alpha2(D14) revealed that mutations affecting the Imp-beta 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-beta 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-alpha2 with a negative regulator of Kelch function.
Gorjánácz
Mátyás
M
Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary.
Török
István
I
Pomozi
István
I
Garab
Gyozo
G
Szlanka
Tamás
T
Kiss
István
I
Mechler
Bernard M
BM
D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D000199:Actins / Q000378:metabolism; D000818:Animals; D004330:Drosophila / Q000196:embryology / Q000235:genetics / Q000648:ultrastructure*; D029721:Drosophila Proteins / Q000032:analysis / Q000235:genetics; D004528:Egg Shell / Q000166:cytology; D005260:Female; D005454:Fluorescence Polarization; D008840:Microfilament Proteins / Q000032:analysis / Q000235:genetics; D008938:Mitosis; D009154:Mutation; D009866:Oogenesis; D010053:Ovary / Q000648:ultrastructure*; D010063:Ovum / Q000166:cytology; D017434:Protein Structure, Tertiary; D028901:alpha Karyopherins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000199:Actins; D029721:Drosophila Proteins; D008840:Microfilament Proteins; D028901:alpha Karyopherins; C087978:karyopherin alpha 2; C080353:kel protein, Drosophila