Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway
Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway
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DOI：
10.1074/jbc.m401066200
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发表时间：
2004-09-03
影响因子：
4.8
通讯作者：
Thumm, M
中科院分区：
文献类型：
--
作者：
Meiling-Wesse, K;Barth, H;Thumm, M
Atg21和Atg18是同源酵母蛋白。Atg18对于Cvt通路和自噬是必不可少的，而缺乏Atg21只会阻断Cvt通路。我们的蛋白酶保护实验现在表明，生长的atg21Delta细胞不能形成含有原氨基肽酶i的Cvt囊泡。定量测量饥饿atg21Delta细胞的自噬率仅为野生型的35%。这表明Atg21在提高自噬保真度方面起着非必要的作用。Atg21的细胞内定位在Atg蛋白中是独一无二的。在含有多个液泡的细胞中，atg21 -黄色荧光蛋白明显定位于液泡连接处的顶点。具有单个液泡的细胞在液泡周围的几个小点上显示大部分蛋白质。这种分布模式让人联想到Vps C类（HOPS）（同型融合和液泡蛋白分选）蛋白复合物。在生长的细胞中，Atg21是at8有效募集到自噬体前结构所必需的。与此一致，at8与脂质磷脂酰乙醇胺的共价键被显著阻滞。脂化at8被认为在自噬体前体的延伸过程中起作用。然而，尽管自噬率降低，at8脂化延迟，但饥饿的atg21Delta ypt7Delta双突变细胞的电镜显示形成了正常大小的自噬体，平均直径为450 nm。
Atg21 and Atg18 are homologue yeast proteins. Whereas Atg18 is essential for the Cvt pathway and autophagy, a lack of Atg21 only blocks the Cvt pathway. Our proteinase protection experiments now demonstrate that growing atg21Delta cells fail to form proaminopeptidase I-containing Cvt vesicles. Quantitative measurement of autophagy in starving atg21Delta cells showed only 35% of the wild-type rate. This suggests that Atg21 plays a nonessential role in improving the fidelity of autophagy. The intracellular localization of Atg21 is unique among the Atg proteins. In cells containing multiple vacuoles, Atg21-yellow fluorescent protein clearly localizes to the vertices of the vacuole junctions. Cells with a single vacuole show most of the protein at few perivacuolar punctae. This distribution pattern is reminiscent to the Vps class C( HOPS) ( homotypic fusion and vacuolar protein sorting) protein complex. In growing cells, Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure. Consistently, the covalent linkage of Atg8 to the lipid phosphatidylethanolamine is significantly retarded. Lipidated Atg8 is supposed to act during the elongation of autophagosome precursors. However, despite the reduced autophagic rate and the retardation of Atg8 lipidation, electron microscopy of starved atg21Delta ypt7Delta double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm.