SdBP associated with Sd and antagonized Sd-Yki-induced overgrowth. (A) Co-immunoprecipation of HA-Sd and 3× Flag-SdBP in S2 cells. The indicated constructs were transfected into S2 cells, followed by immunoprecipitation and western blot analysis with the indicated antibodies. The arrowhead indicates IgG chain. (B) Luciferase activity of a luciferase reporter plasmid with 3× Sd-binding sites and the control vector in S2 cells cotransfected with the indicated constructs. Data were presented as mean ± s.d. (n = 3). (C-F) Gain of function of SdBP induced growth defects and also reduced GMR-Yki-induced overgrowth. Adult eyes expressing GMR-Gal4 (C), GMR-Gal4/UAS-SdBP (D), GMR-Gal4/UAS-Yki (E), and GMR-Gal4/UAS-(SdBP+Yki) (F). (C'-F') BrdU staining of wild-type eye discs (C'), and eye discs expressing UAS-SdBP (D'), UAS-Yki (E') or UAS-(Yki+SdBP) (F') under the control of GMR-Gal4.

SdBP inhibited growth through downregulating the expression of the Hpo pathway target genes. (A-B') Overexpression of SdBP downregulated the expression of the Hpo pathway target genes under the control of act > CD2 > Gal4 (AG4). SdBP overexpression region was marked by Flag (green). DIAP1 (A-A') and Ex (B-B') were stained (blue). Arrowheads indicated the clone regions. (C-F''') Eye discs bearing hpo mutants (C-C'''), hpo mutants overexpressing SdBP (D-D'''), sav mutants (E-E'''), and sav mutants overexpressing SdBP (F-F'''). GFP marked clones, and discs stained with DIAP1 (blue). Arrowheads indicated the clone regions.

TDU domains and PPXY motifs mediated the interaction between SdBP and Sd/Yki. (A) Domain organization of SdBP, Sd and Yki. SdBP contains two TDU domains and three PPXY motifs, and Yki has two WW domains at its C terminus. (B) TDU domain mutations generated in SdBP. Wild-type amino acids were shown in blue, and corresponding mutations were shown in red. (C) Co-immunoprecipation of Sd and the indicated SdBP TDU variants. The arrowhead indicates IgG chain. (D) Myc-Yki and 3× Flag-SdBP interacted with each other in S2 cells. The arrowhead indicates IgG chain. (E) Co-immunoprecipitation between Yki and the indicated SdBP PPXY variants. The arrowhead indicates IgG chain. (F, G) Co-immunoprecipitation assay showed that SdBP interacted with Sd and Yki variants. SdBP TDU variants only disrupted binding between SdBP and Sd, but not Yki (F), and vice versa (G). However, SdBP-(TDU12+PPXY123) bound with neither Sd nor Yki. The indicated constructs in C-G were co-transfected into S2 cells, followed by western blot assay directly or after immunoprecipitation.

TDU domains and PPXY motifs are critical for SdBP function in growth inhibition. (A) Luciferase assay showed that SdBP variants inhibited Sd-Yki activity to different degrees. The indicated plasmids were co-transfected with 3× Sd luciferase reporter, and luciferase activity was measured by standard dual-luciferase protocols. Data were presented as mean ± s.d. (n = 3). (B-G) Scanning electron microscope images of Drosophila compound eyes from the following genotypes: GMR-Gal4 (B), GMR-Gal4/UAS-Yki (C), GMR-Gal4/UAS-(Yki+SdBP) (D), GMR-Gal4/UAS-(Yki+TDU12) (E), GMR-Gal4/UAS-(Yki+PPXY123) (F), and GMR-Gal4/UAS-(Yki+(TDU12+PPXY123)) (G). (H-L) Adult wings of wild-type (H), or wings expressing UAS-SdBP (I), UAS-TDU12 (J), UAS-PPXY123 (K), and UAS-(TDU12+PPXY123) (L) under the control of MS1096. (M) Quantification of relative wing sizes (fold change) of samples indicated in H-L. (N-R') Control wing discs (N) and wing discs expressing UAS-SdBP (O-O'), UAS-TDU12 (P-P'), UAS-PPXY123 (Q-Q') and UAS-(TDU12+PPXY123) (R-R') by hhGal4. SdBP (green) and DIAP1 (blue) were stained. Arrows indicated the P-compartment.

SdBP formed a complex with Sd-Yki. (A) S2 cells expressing the indicated proteins were collected for the two-step immunoprecipitation and analyzed by western blot. Cell lysates were first immunoprecipitated with M2-Flag beads, and then eluted for a second immunoprecipitation with Myc antibody. The arrowheads indicate IgG chain. (B-E''') Confocal images of third-instar wing discs expressing Myc-Yki (B-B'), or Myc-Yki with HA-Sd (C-C') or Myc-Yki with SdBP-GFP (D-D''') or Myc-Yki with HA-Sd and SdBP-GFP (E-E'''). Discs were stained with α-Myc (red), α-HA (magenta) antibodies and DAPI (blue). (F-G') Adult eyes expressing GMR-Gal4/UAS-(Sd+Yki) (F) and GMR-Gal4/UAS-(Sd+Yki+SdBP) (G). BrdU staining of eye discs expressing UAS-(Sd+Yki) (F') and UAS-(Sd+Yki+SdBP) (G') under the control of GMR-Gal4.

SdBP and Yki competed with each other for Sd to control growth. (A-C''') Confocal images of third-instar wing discs bearing hs-FLP clones of sdbp^B mutant (A-A'''), GFP-labeled MARCM clones with Yki overexpression (B-B'''), and GFP-labeled MARCM clones of sdbp^B with Yki overexpression (C-C'''). Cells in clone regions were labeled by SdBP (red), Yki (red), DIAP1 (blue) and GFP (green). (D) GST pull-down assay showed that wild-type SdBP did not affect the stability of Sd-Yki complex while SdBP-PPXY123 competed with Yki for Sd. His-YkiC plus dosage increased wild-type SdBP or SdBP-PPXY123 was pulled down by GST-Sd, followed by western blot using the indicated antibodies. (E) GST pull-down assay showed that Yki also competed with SdBP-PPXY123 for Sd. Wild-type SdBP or SdBP-PPXY123 plus dosage increased His-YkiC were pulled down by GST-Sd, followed by western blot using the indicated antibodies. (F) S2 cells were cotransfected with CFP^N-Sd and YFP^N-Yki. The FRET efficiency was decreased in the presence of SdBP. Data are presented as mean ± s.d. (n ≥ 12). (G) A model of how Hpo signaling regulates the competition between Yki and SdBP for Sd binding. See discussion for details.