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 OGT's 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 OGT's enzymatic activities for cell viability. Using genetic complementation, we found that OGT's 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 OGT's catalytic and noncatalytic activities affect protein abundance, we found that OGT's 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.

中文翻译：

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哺乳动物细胞增殖需要O-GlcNAc转移酶的非催化功能

O-GlcNAc转移酶（OGT）存在于所有哺乳动物细胞类型的细胞核和细胞质中，对细胞增殖至关重要。为什么细胞生长需要OGT尚不清楚。OGT在同一活性位点进行两种酶促反应。一种是糖基化成千上万种不同的蛋白质，另一种是蛋白水解切割涉及基因表达的另一种必需蛋白质。对OGT的无数细胞作用进行反卷积具有挑战性，因为遗传删除具有致命性。补充方法尚未建立。在这里，我们开发了用功能分离变体替代内源性OGT的方法，以研究OGT酶活性对细胞生存力的重要性。利用遗传互补，我们发现OGT' s糖基转移酶功能是细胞生长所必需的，但其蛋白酶功能是必需的。接下来，我们使用互补来构建具有地格隆标签的野生型OGT的细胞系。当OGT降解至非常低的水平时，细胞停止增殖，但仍保持活力。重新添加无催化活性的OGT可以挽救生长。因此，OGT具有细胞增殖所必需的基本非催化作用。通过开发一种量化OGT催化和非催化活性如何影响蛋白质丰度的方法，我们发现OGT的非催化功能通常会影响与其催化功能不同的蛋白质。涉及氧化磷酸化和肌动蛋白细胞骨架的蛋白质尤其受到非催化功能的影响。