Levine, Z. G. | Search bioRxiv

bioRxiv2026

Failure to release from the ER translocon induces 60S ribosome degradation

Ribosome malfunction has toxic consequences, but how mammalian cells select ribosomal subunits for degradation remains poorly understood. Here, we develop a pulse-chase CRISPR screening strategy to identify regulators of ribosome turnover and uncover that loss of protein UFMylation promotes ribosome degradation. Normal UFMylation releases the 60S subunit from the endoplasmic reticulum (ER) translocon. Without UFMylation, single-molecule tracking and optogenetic proximity labeling reveal accumulation of ER-associated 60S subunits and their specific degradation. This translocon-associated large subunit degradation (TLSD) is independent of the lysosome and, in our human cell model, is only active in nondividing, contact-inhibited cells. Degradation requires the poorly characterized protein R3HDM4, which we term TLSD1. Upon UFMylation loss, TLSD1 redistributes to the ER and promotes 60S degradation. These findings identify a cell-state-dependent pathway for clearing ER-retained 60S subunits. We propose translocon release acts as a checkpoint for ribosome function, explaining TLSD1's genetic links to neuronal health and erythropoiesis.

cell biology↗

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↗