Fig. 1. O-fucosylation of Notch. (A) Schematic of EGF repeats in the extracellular domain of Notch. Top: Drosophila Notch, with EGF repeats that conform to a narrow consensus sequence for O-fucosylation [C²XXGG(S/T)C³] shaded pink, and those that conform only to a broader consensus sequence [C²XXX(A/G/S)(S/T)C³] shaded orange. Green bar marks EGF11-12, asterisks mark repeats to which N^Ax mutations map, X marks repeats mutated in this study. Bottom: Consensus O-fucosylation pattern, with EGF repeats shaded according to the fraction of the 15 Notch receptors analyzed that have a broad consensus O-fucose site in that repeat (15/15=black, 0/15=white, intermediate levels of conservation are shaded proportionately gray). (B) Schematic of an EGF domain, illustrating the first two steps in the O-fucosylation pathway. In CHO cells, further elongation occurs; the extent of elongation in Drosophila is unknown. Adapted with permission from Moloney et al. (Moloney et al., 2000a). (C) Amino acid sequences of O-fucose sites mutated in this study. The O-fucose attachment site is underlined; the mutation created is in parenthesis. (D) To characterize O-fucosylation of EGF12, a polypeptide including EGF10-12 and a V5 tag was expressed in S2 cells, partially purified, and assayed as an acceptor substrate for Fringe as described previously (Panin et al., 2002). Fluorography reveals that N-EGF10-12 is a substrate for Fringe, but that the N-EGF10-12f mutant is not. (E) Schematic of part of the wing disc, illustrating Notch signaling at the DV boundary. Serrate (SER) is expressed by dorsal cells and activates Notch (N) in ventral cells; it is blocked from activating Notch in dorsal cells by Fringe. Delta (DL) is expressed by dorsal and ventral cells, but activates Notch principally in dorsal cells, potentiated by Fringe. Notch activation results in the expression of downstream genes, including wg (magenta). (F) Portion of a wing disc, stained for WG protein.