Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation
Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation
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DOI：
10.1080/15548627.2016.1157237
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发表时间：
2016-01-01
期刊：
影响因子：
13.3
通讯作者：
Klionsky, Daniel J.
中科院分区：
文献类型：
--
作者：
Feng, Yuchen;Backues, Steven K.;Klionsky, Daniel J.
巨噬主要是细胞在应激条件下分解自身成分以回收大分子并提供能量的降解过程，巨噬缺陷导致多种疾病。Atg9是从酵母到哺乳动物中保存下来的，是酵母核心巨噬机制中唯一被发现的跨膜蛋白，该机制是形成被称为自噬体的隔离室所必需的。该蛋白在吞噬体组装位点（自噬体前体成核的地方）和可能为吞噬体扩张提供供体膜的外周位点之间进行动态移动。Atg9是一种受Atg1激酶调控的磷酸化蛋白。我们使用细胞培养氨基酸的稳定同位素标记（SILAC）来鉴定该蛋白的磷酸化位点，并在丝氨酸122上鉴定了一个与atg1无关的磷酸化位点。非磷酸化Atg9突变体的自噬活性降低，而拟磷突变体的自噬活性增强。电镜分析表明，不同水平的自噬活性反映了自噬体形成的差异，这与Atg9向PAS的传递有关。最后，这种磷酸化调节Atg9与Atg23和Atg27的相互作用。
Macroautophagy is primarily a degradative process that cells use to break down their own components to recycle macromolecules and provide energy under stress conditions, and defects in macroautophagy lead to a wide range of diseases. Atg9, conserved from yeast to mammals, is the only identified transmembrane protein in the yeast core macroautophagy machinery required for formation of the sequestering compartment termed the autophagosome. This protein undergoes dynamic movement between the phagophore assembly site (PAS), where the autophagosome precursor is nucleated, and peripheral sites that may provide donor membrane for expansion of the phagophore. Atg9 is a phosphoprotein that is regulated by the Atg1 kinase. We used stable isotope labeling by amino acids in cell culture (SILAC) to identify phosphorylation sites on this protein and identified an Atg1-independent phosphorylation site at serine 122. A nonphosphorylatable Atg9 mutant showed decreased autophagy activity, whereas the phosphomimetic mutant enhanced activity. Electron microscopy analysis suggests that the different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Finally, this phosphorylation regulates Atg9 interaction with Atg23 and Atg27.