Impact of Structural Alterations on the Cellular Functions and Interactions of O-GlcNAc Transferase

Abstract

O-GlcNAc Transferase (OGT) is an essential mammalian glycosyltransferase that catalyzes O-GlcNAcylation of serine and threonine residues and cleavage of Host Cell Factor 1. Via these catalytic activities and participation in numerous noncatalytic protein-protein interactions, OGT is involved in myriad cellular processes. In this thesis, I investigate how OGT’s diverse cellular functions are altered by mutations in its catalytic and tetratricopeptide repeat (TPR) domains. In Chapter 2, I collaborate with another graduate student to characterize separation-of-function OGT variants in cells, and we show that OGT's essential biochemical function is Ser/Thr O-GlcNAcylation. In Chapter 3, I use two genetic systems described in Chapter 2 to replace endogenous OGT with a series of OGT TPR truncation variants in order to identify the minimal variant that supports viability. This truncation variant is sufficient to sustain viability despite broadly attenuated Ser/Thr O-GlcNAcylation activity, suggesting maintenance of some essential substrates. In Chapter 4, I characterize the impact of TPR truncation on OGT’s other cellular functions and identify a strong requirement for the N-terminal TPRs in regulating OGT’s subcellular localization, HCF-1 cleavage, and protein-protein interactions. Altogether, the work presented in this thesis reveals that OGT's N-terminal TPRs are intimately involved in all of its cellular activities, but they are not absolutely essential for viability in cell culture. I hypothesize that the minimal viable truncation variant that I have discovered will facilitate future identification of OGT’s essential cellular substrates and interactors.