title: Multiple O-glucosylation sites on Notch function as a buffer against temperature-dependent loss of signaling.
abstract: Mutations in Drosophila rumi result in a temperature-sensitive loss of Notch signaling. Rumi is a protein O-glucosyltransferase that adds glucose to EGF repeats with a C-X-S-X-P-C consensus sequence. Eighteen of the 36 EGF repeats in the Drosophila Notch receptor contain the consensus O-glucosylation motif. However, the contribution of individual O-glucose residues on Notch to the regulation of Notch signaling is not known. To address this issue, we carried out a mutational analysis of these glucosylation sites and determined their effects on Notch activity in vivo. Our results indicate that even though no single O-glucose mutation causes a significant decrease in Notch activity, all of the glucose residues on Notch contribute in additive and/or redundant fashions to maintain robust signaling, especially at higher temperatures. O-glucose motifs in and around the ligand-binding EGF repeats play a more important role than those in other EGF repeats of Notch. However, a single O-glucose mutation in EGF12 can be compensated by other O-glucose residues in neighboring EGF repeats. Moreover, timecourse cell aggregation experiments using a rumi null cell line indicate that a complete lack of Rumi does not affect Notch-Delta binding at high temperature. In addition, rumi fully suppresses the gain-of-function phenotype of a ligand-independent mutant form of Notch. Our data suggest that, at physiological levels of Notch, the combined effects of multiple O-glucose residues on this receptor allow productive S2 cleavage at high temperatures and thereby serve as a buffer against temperature-dependent loss of Notch signaling.
authors.affiliation: Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Leonardi
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Fernandez-Valdivia
authors.forename: Yi-Dong
authors.initials: YD
authors.lastname: Li
authors.forename: Amanda A
authors.initials: AA
authors.lastname: Simcox
authors.forename: Hamed
authors.initials: H
authors.lastname: Jafar-Nejad
mesh_terms: D000818:Animals; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D049750:Genome, Insect; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013696:Temperature; D019076:Transgenes
chemical_list: D029721:Drosophila Proteins; C497759:N protein, Drosophila; D051880:Receptors, Notch; D005964:Glucosyltransferases; C527374:Rumi protein, Drosophila; D005947:Glucose