ABSTRACT

Macroautophagy (autophagy) is driven by the coordinated actions of core autophagy-related (Atg) proteins. Atg8, the core Atg protein generally considered acting most downstream, has recently been shown to interact with other core Atg proteins via their Atg8-family-interacting motifs (AIMs). However, the extent, functional consequence, and evolutionary conservation of such interactions remain inadequately understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg38, a subunit of the phosphatidylinositol 3-kinase (PtdIns3K) complex I, interacts with Atg8 via an AIM, which is highly conserved in Atg38 proteins of fission yeast species, but not conserved in Atg38 proteins of other species. This interaction recruits Atg38 to Atg8 on the phagophore assembly site (PAS) and consequently enhances PAS accumulation of the PtdIns3K complex I and Atg proteins acting downstream of the PtdIns3K complex I, including Atg8. The disruption of the Atg38-Atg8 interaction leads to the reduction of autophagosome size and autophagic flux. Remarkably, the loss of this interaction can be compensated by an artificial Atg14-Atg8 interaction. Our findings demonstrate that the Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop between Atg8 and the PtdIns3K complex I to promote efficient autophagosome formation, underscore the prevalence and diversity of AIM-mediated connections within the autophagic machinery, and reveal unforeseen flexibility of such connections.

Abbreviations: AIM: Atg8-family-interacting motif; AP-MS: affinity purification coupled with mass spectrometry; Atg: autophagy-related; FLIP: fluorescence loss in photobleaching; PAS: phagophore assembly site; PB: piggyBac; PE: phosphatidylethanolamine; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate.

摘要

巨自噬 (autophagy) 是由核心自噬相关 (Atg) 蛋白的协同作用驱动的。Atg8 是通常被认为作用最下游的核心 Atg 蛋白，最近已显示通过其 Atg8 家族相互作用基序 (AIM) 与其他核心 Atg 蛋白相互作用。然而，对这种相互作用的程度、功能后果和进化保守性仍知之甚少。在这里，我们表明，在裂殖酵母中，粟酒裂殖酵母, Atg38 是磷脂酰肌醇 3-激酶 (PtdIns3K) 复合物 I 的一个亚基，通过 AIM 与 Atg8 相互作用，AIM 在裂变酵母物种的 Atg38 蛋白中高度保守，但在其他物种的 Atg38 蛋白中不保守。这种相互作用将 Atg38 募集到吞噬细胞组装位点 (PAS) 上的 Atg8，从而增强 PtdIns3K 复合物 I 和作用于 PtdIns3K 复合物 I 下游的 Atg 蛋白（包括 Atg8）的 PAS 积累。Atg38-Atg8 相互作用的破坏导致自噬体大小和自噬通量的减少。值得注意的是，这种相互作用的损失可以通过人工 Atg14-Atg8 相互作用来补偿。

缩写：AIM：Atg8-family-interacting 母题；AP-MS：亲和纯化结合质谱；Atg：自噬相关；FLIP：光漂白中的荧光损失；PAS：吞噬细胞组装位点；PB：piggyBac；PE：磷脂酰乙醇胺；PtdIns3K：磷脂酰肌醇 3-激酶；PtdIns3P：3-磷酸磷脂酰肌醇。