用于分析酵母线粒体中蛋白质磷酸化的先进工具。

Advanced tools for the analysis of protein phosphorylation in yeast mitochondria.

作者信息

Walter Corvin, Gonczarowska-Jorge Humberto, Sickmann Albert, Zahedi René P, Meisinger Chris, Schmidt Oliver

机构信息

Institute of Biochemistry and Molecular Biology, ZBMZ, Faculty of Medicine, University of Freiburg, Germany; Faculty of Biology, University of Freiburg, Germany.

Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany; CAPES Foundation, Ministry of Education of Brazil, Brasília, DF 70040-020, Brazil.

出版信息

Anal Biochem. 2018 Aug 1;554:23-27. doi: 10.1016/j.ab.2018.05.022. Epub 2018 May 24.

DOI:10.1016/j.ab.2018.05.022

PMID:29803787

Abstract

The biochemical analysis of protein phosphorylation in mitochondria lags behind that of cytosolic signaling events. One reason is the poor stability of many phosphorylation sites during common isolation procedures for mitochondria. We present here an optimized, fast protocol for the purification of yeast mitochondria that greatly increases recovery of phosphorylated mitochondrial proteins. Moreover, we describe improved protocols for the biochemical analysis of mitochondrial protein phosphorylation by Zn-Phos-tag electrophoresis under both denaturing and - for the first time - native conditions, and demonstrate that they outperform previously applied methods.

摘要

线粒体中蛋白质磷酸化的生化分析落后于胞质信号转导事件的分析。一个原因是在常用的线粒体分离程序中，许多磷酸化位点的稳定性较差。我们在此提出一种优化的、快速的酵母线粒体纯化方案，该方案大大提高了磷酸化线粒体蛋白的回收率。此外，我们描述了在变性条件下以及首次在天然条件下通过锌 - 磷光标记电泳对线粒体蛋白磷酸化进行生化分析的改进方案，并证明它们优于以前应用的方法。