The Protein O-glucosyltransferase Rumi Modifies Eyes Shut to Promote Rhabdomere Separation in Drosophila

Abstract

The protein O-glucosyltransferase Rumi/POGLUT1 regulates Drosophila Notch signaling by adding O-glucose residues to the Notch extracellular domain. Rumi has other predicted targets including Crumbs (Crb) and Eyes shut (Eys), both of which are involved in photoreceptor development. However, whether Rumi is required for the function of Crb and Eys remains unknown. Here we report that in the absence of Rumi or its enzymatic activity, several rhabdomeres in each ommatidium fail to separate from one another in a Notch-independent manner. Mass spectral analysis indicates the presence of O-glucose on Crb and Eys. However, mutating all O-glucosylation sites in a crb knock-in allele does not cause rhabdomere attachment, ruling out Crb as a biologically-relevant Rumi target in this process. In contrast, eys and rumi exhibit a dosage-sensitive genetic interaction. In addition, although in wild-type ommatidia most of the Eys protein is found in the inter-rhabdomeral space (IRS), in rumi mutants a significant fraction of Eys remains in the photoreceptor cells. The intracellular accumulation of Eys and the IRS defect worsen in rumi mutants raised at a higher temperature, and are accompanied by a ∼50% decrease in the total level of Eys. Moreover, removing one copy of an endoplasmic reticulum chaperone enhances the rhabdomere attachment in rumi mutant animals. Altogether, our data suggest that O-glucosylation of Eys by Rumi ensures rhabdomere separation by promoting proper Eys folding and stability in a critical time window during the mid-pupal stage. Human EYS, which is mutated in patients with autosomal recessive retinitis pigmentosa, also harbors multiple Rumi target sites. Therefore, the role of O-glucose in regulating Eys may be conserved.

Author Summary

Glycosylation (addition of sugars to proteins and other organic molecules) is important for protein function and animal development. Each form of glycosylation is usually present on multiple proteins. Therefore, a major challenge in understanding the role of sugars in animal development is to identify which protein(s) modified by a specific sugar require the sugar modification for proper functionality. We have previously shown that an enzyme called Rumi adds glucose molecules to an important cell surface receptor called Notch, and that glucose plays a key role in the function of Notch both in fruit flies and in mammals. Using fruit flies, we have now identified a new Rumi target called “Eyes shut”, a secreted protein with a critical role in the optical isolation of neighboring photoreceptors in the fly eye. Our data suggest that glucose molecules on Eyes shut promote its folding and stability in a critical time window during eye development. Mutations in human Eyes shut result in a devastating form of retinal degeneration and loss of vision. Since human Eyes shut is also predicted to harbor glucose molecules, our work provides a framework to explore the role of sugar modifications in the biology of a human disease protein.