An APEX2-based proximity-dependent biotinylation assay with temporal specificity to study protein interactions during autophagy in the yeast Saccharomyces cerevisiae

被引:1
作者
Filali-Mouncef, Yasmina [1 ]
Leytens, Alexandre [2 ]
Duarte, Prado Vargas [3 ]
Zampieri, Mattia [4 ]
Dengjel, Jorn [2 ]
Reggiori, Fulvio [1 ,3 ,5 ]
机构
[1] Univ Groningen, Univ Med Ctr Groningen, Dept Biomed Sci Cells & Syst, A Deusinglaan 1, NL-9713 AV Groningen, Netherlands
[2] Univ Fribourg, Dept Biol, Chemin Musee 10, CH-1700 Fribourg, Switzerland
[3] Aarhus Univ, Dept Biomed, Aarhus, Denmark
[4] Univ Basel, Dept Biomed, Basel, Switzerland
[5] Aarhus Univ, Aarhus Inst Adv Studies AIAS, Aarhus, Denmark
基金
瑞士国家科学基金会;
关键词
Atg proteins; Atg8; Atg9; mass spectrometry; proteomics; proximity labeling; RECRUITS ATG9; SITES; VECTORS;
D O I
10.1080/15548627.2024.2366749
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Autophagosome biogenesis is a complex process orchestrated by dynamic interactions between Atg (autophagy-related) proteins and characterized by the turnover of specific cargoes, which can differ over time and depending on how autophagy is stimulated. Proteomic analyses are central to uncover protein-protein interaction networks and when combined with proximity-dependent biotinylation or proximity labeling (PL) approaches, they also permit to detect transient and weak interactions. However, current PL procedures for yeast Saccharomyces cerevisiae, one of the leading models for the study of autophagy, do not allow to keep temporal specificity and thus identify interactions and cargoes at a precise time point upon autophagy induction. Here, we present a new ascorbate peroxidase 2 (APEX2)-based PL protocol adapted to yeast that preserves temporal specificity and allows uncovering neighbor proteins by either western blot or proteomics. As a proof of concept, we applied this new method to identify Atg8 and Atg9 interactors and detected known binding partners as well as potential uncharacterized ones in rich and nitrogen starvation conditions. Also, as a proof of concept, we confirmed the spatial proximity interaction between Atg8 and Faa1. We believe that this protocol will be a new important experimental tool for all those researchers studying the mechanism and roles of autophagy in yeast, but also other cellular pathways in this model organism.
引用
收藏
页码:2323 / 2337
页数:15
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