FIGURE 1.

O-Fucosyltransferase activity of O-fut1^{R245A knock-in} is negligible in vivo. A, schematic of the O-Fut1 protein and its mutant derivatives. The GDP-fucose-binding motif (GXHXR(R/H)) and ER retention signal (HEEL) are shown in magenta and green, respectively. Top, middle, and bottom bars represent wild-type, O-Fut1^R245A, and O-Fut1^4R6, respectively. O-Fut1^R245A has an amino acid substitution (Arg to Ala) at the 245th amino acid that disrupts its O-fucosyltransferase activity. O-Fut1^4R6 carries a nonsense mutation that introduces a premature stop codon at the 133rd amino acid. Black bar corresponds to 100 amino acids. B and C, wild-type (B) and O-fut1^{R245A knock-in} homozygote (C) wing discs of third-instar larvae were stained with an anti-Wg antibody. The expression of wg along the D/V boundary, which depends on N signaling, is indicated by an arrowhead in B. D, dorsal compartment; V, ventral compartment. D–E′, UAS-fng was misexpressed under the control of dpp-Gal4 along the anterior-posterior boundary of wild-type (D and D′) and O-fut1^{R245A knock-in} homozygote wing discs (E and E′). These wing discs were stained with an anti-Wg antibody (magenta in D and E and white in D′ and E′). The expression pattern of dpp-Gal4 was monitored by detecting the UAS-GFP expression using an anti-GFP antibody (green in D and E). D′ and E′ are single channel images of the magenta (anti-Wg staining) in D and E, respectively. The ectopic expression of wg is indicated by an arrowhead in D′. Scale bars, B–E′ are 100 μm.