PDF(Pubmed)
Abstract：
Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the preautophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.
摘要：
自噬是一种选择性降解过氧化物酶体的自噬，可分为巨噬细胞自噬或微自噬。在巨噬期间，单个过氧化物酶体被pexophagosome隔离并运输到液泡进行降解，而在微噬菌体中，过氧化物酶体被分隔的液泡直接吞噬。迄今为止,一些自噬所需的自噬相关基因(ATGs)已通过基于平板的试验进行鉴定,这些试验主要在微自噬诱导条件下进行.这里,我们开发了一种新的高通量筛选系统,该系统使用荧光激活细胞分选(FACS)来鉴定巨噬细胞吞噬所需的基因.使用这个系统，我们发现了KpATG14,由于技术限制,该基因以前无法在甲基营养型酵母Komagataellaphafii中鉴定.显微镜和免疫印迹分析发现，巨噬和微噬都需要KpAtg14。我们还发现，KpAtg14对于自噬前结构（PAS）的下游因子KpAtg5的募集是必需的，因此，大量自噬。我们预计我们的检测方法将用于鉴定巨噬细胞所需的新基因，导致更好地理解现有的两种类型的过氧化物酶体自噬降解途径的生理意义。
