Gómez-Sánchez R, Sánchez-Wandelmer J, Reggiori F
University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Methods Enzymol. 2017;588:323-365. doi: 10.1016/bs.mie.2016.09.085. Epub 2016 Nov 21.
The budding yeast Saccharomyces cerevisiae is a powerful and versatile model organism for studying multiple aspects of the biology of eukaryotic cells, including the molecular principles underlying autophagy. One of the unique advantages of this unicellular system is its amenability to genetic and biochemical approaches, which had a pivotal role in the discovery and characterization of most of the autophagy-related (Atg) proteins, the central players of autophagy. The relevance of investigating autophagy in this cell model lies in the high conservation of this pathway among eukaryotes, i.e., most of the yeast Atg proteins possess one or more mammalian orthologs. In addition to the experimental advantages, a very large collection of reagents keeps S. cerevisiae in a leading position for the study of the molecular mechanism and regulation of autophagy. In this chapter, we describe fluorescence microscopy and biochemical methods that allow to monitor in vivo the assembly the of Atg machinery, a key step of autophagy. These approaches can be very useful to those researchers that would like to assess the progression of the autophagosomal precursor structure formation under various conditions, in the presence of specific Atg protein mutants or in the absence of other factors.
出芽酵母酿酒酵母是研究真核细胞生物学多个方面的强大且通用的模式生物，包括自噬的分子原理。这个单细胞系统的独特优势之一是它适合采用遗传学和生物化学方法，这些方法在发现和鉴定大多数自噬相关（Atg）蛋白（自噬的核心参与者）方面发挥了关键作用。在这个细胞模型中研究自噬的意义在于该途径在真核生物中具有高度保守性，即大多数酵母Atg蛋白拥有一个或多个哺乳动物直系同源物。除了实验优势外，大量的试剂使酿酒酵母在自噬分子机制和调控研究中处于领先地位。在本章中，我们描述了荧光显微镜和生化方法，这些方法可以在体内监测Atg机制的组装，这是自噬的关键步骤。对于那些想要评估在各种条件下、存在特定Atg蛋白突变体或不存在其他因素时自噬体前体结构形成进程的研究人员来说，这些方法可能非常有用。
