Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies, CNR, Bari, Italy.
Department of Biosciences, Biotechnology and Biopharmaceutics, University of Bari "A. Moro", Bari, Italy.
Methods Mol Biol. 2021;2276:87-102. doi: 10.1007/978-1-0716-1266-8_6.
Mitochondrial retrograde signaling is a mitochondria-to-nucleus communication pathway, conserved from yeast to humans, by which dysfunctional mitochondria relay signals that lead to cell stress adaptation in physiopathological conditions via changes in nuclear gene expression. The most comprehensive picture of components and regulation of retrograde signaling has been obtained in Saccharomyces cerevisiae, where retrograde-target gene expression is regulated by RTG genes. In this chapter, we describe methods to measure mitochondrial retrograde pathway activation at the level of mRNA and protein products in yeast model systems, including cell suspensions and microcolonies. In particular, we will focus on three major procedures: mRNA levels of RTG-target genes, such as those encoding for peroxisomal citrate synthase (CIT2), aconitase, and NAD-specific isocitrate dehydrogenase subunit 1 by real-time PCR; expression analysis of CIT2-gene protein product (Cit2p-GFP) by Western blot and fluorescence microscopy; the phosphorylation status of transcriptional factor Rtg1/3p which controls RTG-target gene transcription.
线粒体逆行信号转导是一种从酵母到人都保守的线粒体到核的通讯途径，通过该途径，功能失调的线粒体传递信号，导致细胞在生理病理条件下通过核基因表达的变化适应应激。在酿酒酵母中，逆行靶向基因表达受 RTG 基因调控，获得了逆行信号转导的最全面的成分和调控图谱。在本章中，我们描述了在酵母模型系统中测量 mRNA 和蛋白质产物水平的线粒体逆行途径激活的方法，包括细胞悬浮液和微菌落。特别地，我们将集中讨论三个主要程序：实时 PCR 测定 RTG 靶向基因（如编码过氧化物酶体柠檬酸合酶（CIT2）、顺乌头酸酶和 NAD 特异性异柠檬酸脱氢酶亚基 1 的基因）的 mRNA 水平；通过 Western blot 和荧光显微镜分析 CIT2 基因蛋白产物（Cit2p-GFP）的表达；转录因子 Rtg1/3p 的磷酸化状态，该因子控制 RTG 靶向基因转录。
