研究酵母中的蛋白质泛素化

Studying Protein Ubiquitylation in Yeast.

作者信息

Hovsepian Junie, Becuwe Michel, Kleifeld Oded, Glickman Michael H, Léon Sébastien

机构信息

Institut Jacques Monod, UMR 7592 CNRS/Univ. Paris Diderot, Sorbonne Paris Cité, Paris, France.

Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA, 02115, USA.

出版信息

Methods Mol Biol. 2016;1449:117-42. doi: 10.1007/978-1-4939-3756-1_5.

DOI:10.1007/978-1-4939-3756-1_5

PMID:27613031

Abstract

Ubiquitylation is a reversible posttranslational modification that is critical for most, if not all, cellular processes and essential for viability. Ubiquitin conjugates to substrate proteins either as a single moiety (monoubiquitylation) or as polymers composed of ubiquitin molecules linked to each other with various topologies and structures (polyubiquitylation). This contributes to an elaborate ubiquitin code that is decrypted by specific ubiquitin-binding proteins. Indeed, these different types of ubiquitylation have different functional outcomes, notably affecting the stability of the substrate, its interactions, its activity, or its subcellular localization. In this chapter, we describe protocols to determine whether a protein is ubiquitylated, to identify the site that is ubiquitylated, and provide direction to study the topology of the ubiquitin modification, in the yeast Saccharomyces cerevisiae.

摘要

泛素化是一种可逆的翻译后修饰，对大多数（即便不是全部）细胞过程至关重要，且对细胞存活必不可少。泛素与底物蛋白结合，既可以作为单个部分（单泛素化），也可以作为由泛素分子以各种拓扑结构和形式相互连接而成的聚合物（多泛素化）。这促成了一种精细的泛素密码，由特定的泛素结合蛋白进行解密。实际上，这些不同类型的泛素化具有不同的功能结果，尤其会影响底物的稳定性、相互作用、活性或亚细胞定位。在本章中，我们描述了在酿酒酵母中确定蛋白质是否被泛素化、识别被泛素化的位点以及为研究泛素修饰的拓扑结构提供指导的实验方案。