Valdbjørn Kanne Jakob, Reggiori Fulvio
Department of Biomedicine, Aarhus University, Aarhus C, Denmark.
Autophagy Rep. 2025 May 31;4(1):2511724. doi: 10.1080/27694127.2025.2511724. eCollection 2025.
Monitoring the delivery of single proteins and protein complexes to the vacuole by autophagy or other processes in yeast mainly relies on western blot or fluorescence microscopy analyses using endogenous tagging of the protein of interest with GFP. However, these approaches are semi-quantitative and next to impossible with proteins of low abundancy because of the insensitive nature of the methods. Here, we describe the creation of a new PCR-based integration cassette to endogenously tag specific proteins with the truncated version of the vacuolar phosphatase Pho8. The vacuolar activation of Pho8 allows the quantitative measurement of vacuolar delivery using a colorimetric enzymatic assay. This approach has the advantages of a more quantitative interpretation of data and relies on the appearance of a signal rather than its disappearance. As a proof-of-principle, we examined the vacuolar delivery of known cargoes of bulk autophagy and endocytosis. This new system will be of great value to the whole community working within the field of autophagy and other transport pathways to the vacuole.
监测酵母中通过自噬或其他过程将单个蛋白质和蛋白质复合物输送到液泡主要依赖于蛋白质免疫印迹法或荧光显微镜分析，这些分析使用绿色荧光蛋白（GFP）对目标蛋白质进行内源性标记。然而，这些方法是半定量的，并且对于低丰度蛋白质几乎无法实现，因为这些方法本质上不灵敏。在这里，我们描述了一种基于PCR的新整合盒的构建，用于用液泡磷酸酶Pho8的截短版本对内源特定蛋白质进行标记。Pho8在液泡中的激活使得能够使用比色酶法对液泡输送进行定量测量。这种方法具有对数据进行更定量解释的优点，并且依赖于信号的出现而非消失。作为原理验证，我们检测了已知的批量自噬和内吞作用货物的液泡输送。这个新系统对于在自噬和其他向液泡的运输途径领域工作 的整个群体将具有巨大价值。
