Joiner, C. M. | Search bioRxiv

bioRxiv2020

Protein substrates engage the lumen of O-GlcNAc transferase's tetratricopeptide repeat domain in different ways

Glycosylation of nuclear and cytoplasmic proteins is an essential post-translational modification in mammals. O-GlcNAc transferase (OGT), the sole enzyme responsible for this modification, glycosylates over a thousand unique nuclear and cytoplasmic substrates. How OGT selects its substrates is a fundamental question that must be answered to understand OGTs unusual biology. OGT contains a long tetratricopeptide repeat (TPR) domain that has been implicated in substrate selection, but there is almost no information about how changes to this domain affect glycosylation of individual substrates. Here, we used proteome-wide glycosylation profiling and probed glycosylation of selected purified substrates to show that asparagine and aspartate ladders that extend the full length of OGTs TPR lumen control substrate glycosylation. We also found that substrates with glycosylation sites close to the C-terminus bypass lumenal binding. Our findings demonstrate that substrates can engage OGT in a variety of different ways for glycosylation.

biochemistry↗

bioRxiv2020

Mammalian Cell Proliferation Requires Noncatalytic Functions of O-GlcNAc Transferase

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGTs myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGTs enzymatic activities for cell viability. Using genetic complementation, we found that OGTs glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically-inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGTs catalytic and noncatalytic activities affect protein abundance, we found that OGTs noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology. SignificanceMammalian cells contain only one glycosyltransferase, OGT, that operates in the nucleus and cytoplasm rather than the secretory pathway. OGT is required for cell proliferation, but a basic unanswered question is what OGT functions are essential. This question is challenging to address because OGT has thousands of glycosylation substrates, two different enzymatic activities, and a large number of binding partners. Here, by establishing genetic tools to replace endogenous OGT with variants that preserve only a subset of its activities, we show that only a low level of glycosylation activity is required to maintain cell viability; however, cell proliferation requires noncatalytic OGT function(s). The ability to replace OGT with variants provides a path to identifying its essential substrates and binding partners.

biochemistry↗