Two Distinct E3 Ubiquitin Ligases Have Complementary Functions in the Regulation of Delta and Serrate Signaling in Drosophila (original) (raw)
Figure 1
Molecular and Genetic Characterization of D-mib Mutations
(A) Molecular map of the D-mib locus showing the position of the P-element inserted into the 5′ untranslated region (allele D-mib1) and the 13.6 kb deletion that removes the D-mib and the RpS31 genes (allele D-mib2). Transcribed regions are indicated with arrows, and exons are indicated with boxes. Open reading frames are shown in black.
(B) Domain composition of D-mib and D. rerio Mib. Both proteins show identical domain organization. D-mib has an N-terminal ZZ zinc finger flanked on either side by a Mib/HERC2 (M-H) domain, followed by two Mib repeats, six ankyrin repeats, two atypical RING domains, and a C-terminal protypical RING that has been associated with catalytic E3 ubiquitin ligase activity. The D-mib3 mutant allele is predicted to produce a truncated protein devoid of E3 ubiquitin ligase activity whereas the D-mib4 protein carries a mutation at a conserved position in the second Mib repeat.
(C and C′) Western blot analysis of D-mib (C). The endogenous D-mib protein (predicted size: 130 kDa) was detected in S2 cells (lane 2) and in imaginal discs from wild-type larvae (lane 3) but was not detectable in homozygous D-mib1 (lane 4) and D-mib1/D-mib3 (lane 5) third instar larvae. The D-mib protein produced in transfected S2 cells from the cDNA used in this study (lane 1) runs exactly as endogenous D-mib (lane 2). Panel C′ shows a Red Ponceau staining of the gel with the same protein samples as in panel C.
(D–H) Wings from wild-type (D), D-mib1 (E), SerRX82/Serrev6.1 (F), D-mib2/D-mib4 (G), and UAS-D-mib2/+; D-mib1/D-mib2 flies (H). D-mib (E) and Ser (F) mutant flies showed similar wing loss phenotypes. The D-mib mutant phenotype could be almost fully rescued by a leaky UAS-D-mib transgene (H). (D′) and (G′) show high magnification views of (D) and (G), respectively, to show that D-mib2/D-mib4 mutant flies (G′) exhibited ectopic sensilla (arrowheads) along vein L3.
(I–N) Nota (I–K) and legs (L–N) from wild-type (I and L), D-mib1 (J and M), and SerRX82/Serrev6.1 (K and N) flies. D-mib mutant flies showed a weak neurogenic phenotype (J) that was not observed in Ser mutant flies (K). Ectopic sensory organs in D-mib mutant flies developed from ectopic sensory organ precursor cells (not shown). D-mib (M) and Ser (N) mutant legs also showed distinct growth and/or elongation defects. Arrows in (J) show ectopic macrochaetes. Arrows in (L–N) indicate the joints. Ti, tibia; t1 to t5, tarsal segments 1 to 5.