Amino acids in motifs 2 are required for Takeout function.

(A) Conserved motifs 1 (yellow) and 2 (red) are exposed at the bottom of the protein in the D. mel model (A), separated by helix 2 (green). Residues that are at the bottom of the protein and predicted to be exposed were considered for site directed mutagenesis in D. mel TO. (B) Alignment of Motif 2 in all tested To homologs using PRALINE is shown. Conservation scores generated by the program are color coded, and a conservation index is indicated underneath each residue. Note that CG16820 shows several deviations from the consensus in this region. Residues colored in yellow were mutated to Alanine, including a Phenylalanine at position 258 that was conserved in all sequences, creating TO-mut. (C) TO-mut only partially rescues the takeout courtship defect. Graph shows the courtship index CI (fraction of time males spend courting during the observation period) ± SEM of the indicated genotypes. The Lsp-2-Gal4 fat body driver was used to express TO-mut in a fru4to1/to1 mutant background. Male flies heterozygous for to1 show robust courtship (a, green, CI 0.96±0.01). to1 homozygous flies with a fru4 heterozygous genetic background show severely reduced courtship (b, yellow, 0.52±0.02). While a To wildtype transgene rescues the behavior (c, light blue, CI 0.91±0.02), expression of TO-mut only shows partial rescue (d, blue, 0.76±0.03). Data were analyzed by ANOVA followed by Bonferroni multiple comparisons (N = 20).