ABSTRACT

Macroautophagy/autophagy is a key catabolic process in which different cellular components are sequestered inside double-membrane vesicles called autophagosomes for subsequent degradation. In yeast, autophagosome formation occurs at the phagophore assembly site (PAS), a specific perivacuolar location that works as an organizing center for the recruitment of different autophagy-related (Atg) proteins. How the PAS is localized to the vacuolar periphery is not well understood. Here we show that the vacuolar membrane protein Vac8 is required for correct vacuolar localization of the PAS. We provide evidence that Vac8 anchors the PAS to the vacuolar membrane by binding Atg13 and recruiting the Atg1 initiation complex. VAC8 deletion or mislocalization of the protein reduce autophagy activity, highlighting the importance of both the PAS and the correct vacuolar localization of the Atg1 initiation complex for efficient and robust autophagy.

Abbreviations: AID: auxin-inducible degradation; Atg: autophagy-related; Cvt: cytoplasm-to-vacuole targeting; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; GFP: green fluorescent protein; IAA: 3-indole acetic acid; PAS: phagophore assembly site; RFP: red fluorescent protein

摘要

巨自噬/自噬是一个关键的分解代谢过程，其中不同的细胞成分被隔离在称为自噬体的双膜囊泡内，以便随后降解。在酵母中，自噬体形成发生在吞噬细胞组装位点 (PAS)，这是一个特定的液泡周围位置，可作为募集不同自噬相关 (Atg) 蛋白的组织中心。PAS 如何定位于液泡周边尚不清楚。在这里，我们表明液泡膜蛋白 Vac8 是 PAS 正确液泡定位所必需的。我们提供的证据表明，Vac8 通过结合 Atg13 和招募 Atg1 起始复合物将 PAS 锚定到液泡膜上。VAC8 蛋白质的缺失或错误定位会降低自噬活性，突出了 PAS 和 Atg1 起始复合物的正确液泡定位对于高效和稳健的自噬的重要性。

缩写： AID：生长素诱导降解；Atg：自噬相关；Cvt：细胞质到液泡靶向；DMSO：二甲亚砜；ER：内质网；GFP：绿色荧光蛋白；IAA：3-吲哚乙酸；PAS：吞噬细胞组装位点；RFP：红色荧光蛋白