SUMMARYProtein O-glucosylation is a conserved post-translational modification that occurs on epidermal growth factor-like (EGF) repeats harboring the C 1 -X-S-X-P-C 2 consensus sequence.
The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14–20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16–20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16–20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16–20 of Notch.
Telomerase is the ribonucleoprotein complex that adds telomeric repeats to the ends of chromosomes. Its protein subunit TERT is highly conserved among eukaryotes, whereas the RNA subunit varies greatly in size and sequence, hindering the identification of telomerase RNAs in some important model organisms. Here we report the identification and functional characterization of TER1, the telomerase RNA component from fission yeast Schizosaccharomyces pombe. Deletion of ter1+ caused progressive shortening of telomeres and cellular senescence followed by chromosome circularization. Interactions between Est1 and Trt1, the two known protein components of fission yeast telomerase, were dependent on TER1, supporting its role as a scaffold for the assembly of protein subunits. Using a series of template mutations, we show that translocation or dissociation site variability and template-primer slippage account for the sequence heterogeneity of fission yeast telomeres.
SUMMARYMutations 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.
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