Date of Graduation

5-2015

Document Type

Dissertation (PhD)

Program Affiliation

Genes and Development

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Hamed Jafar-Nejad

Committee Member

William Mattox

Committee Member

Michael Galko

Committee Member

Kartik Venkatachalam

Committee Member

Sheng Zhang

Committee Member

Hugo Bellen

Abstract

Rumi is a protein O-glucosyltransferase that adds the sugar O-glucose onto the serine in the target sequence C-S-X-S-(P/A)-C found within properly folded EGF repeats. It was first discovered to modify the Drosophila Notch extracellular domain and to be required for Notch signaling in a temperature dependent manner, but other targets of Rumi remained unknown. Several other proteins in the Drosophila proteome harbor multiple consensus sequence highly predictive of O-glucose, including the transmembrane protein Crumbs and the secreted protein Eyes shut (Eys). Both of these proteins are required for proper eye development and mutations in their human homologs cause a blindness disorder named retinitis pigmentosa. Therefore, we sought to determine whether Rumi plays a role in photoreceptor development. We found that rumi–/– animals have defects in photoreceptor spacing in which many neighboring rhabdomeres are attached. This phenotype cannot be explained by the loss of O-glucose on Notch or Crumbs. However, eys genetically interacts with rumi, and in rumi–/– animals at the start of rhabdomere separation, Eys accumulates intracellularly and decreased levels of Eys reach the extracellular space. Overexpressing a mutant Eys transgene which contains no intact O-glucosylation sites also results in intracellular accumulation of Eys, suggesting that loss of O-glucose from Eys is the cause. Additionally, both the intracellular accumulation and the rhabdomere attachment defect grow more severe at higher temperatures, and Eys degrades at higher temperatures in rumi–/–. In addition, removing one copy of the chaperone Hsc70-3 enhances the rumi–/– phenotype. Together, these data suggest that loss of O-glucose from Eys causes a defect in its proper folding, which leads to decreased Eys reaching the extracellular space and therefore a failure in full separation of the rhabdomeres.

Keywords

Rumi, glycosylation, O-glucosylation, photoreceptor, Eyes shut, Eys

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