ABSTRACT

Macroautophagy/autophagy delivers cytoplasmic cargo to lysosomes for degradation. In yeast, the single Atg8 protein plays a role in the formation of autophagosomes whereas in mammalian cells there are five to seven paralogs, referred to as mammalian Atg8s (mAtg8s: GABARAP, GABARAPL1, GABARAPL2, LC3A, LC3B, LC3B2 and LC3C) with incompletely defined functions. Here we show that a subset of mAtg8s directly control lysosomal biogenesis. This occurs at the level of TFEB, the principal regulator of the lysosomal transcriptional program. mAtg8s promote TFEB’s nuclear translocation in response to stimuli such as starvation. GABARAP interacts directly with TFEB, whereas RNA-Seq analyses reveal that knockout of six genes encoding mAtg8s, or a triple knockout of the genes encoding all GABARAPs, diminishes the TFEB transcriptional program. We furthermore show that GABARAPs in cooperation with other proteins, IRGM, a factor implicated in tuberculosis and Crohn disease, and STX17, are required during starvation for optimal inhibition of MTOR, an upstream kinase of TFEB, and activation of the PPP3/calcineurin phosphatase that dephosphorylates TFEB, thus promoting its nuclear translocation. In conclusion, mAtg8s, IRGM and STX17 control lysosomal biogenesis by their combined or individual effects on MTOR, TFEB, and PPP3/calcineurin, independently of their roles in the formation of autophagosomal membranes.

Abbreviations: AMPK: AMP-activated protein kinase; IRGM: immunity related GTPase M; mAtg8s: mammalian Atg8 proteins; MTOR: mechanistic target of rapamycin kinase; PPP3CB: protein phosphatase 3 catalytic subunit beta; RRAGA: Ras related GTP binding A.; STX17: syntaxin 17; ULK1: unc-51 like autophagy activating kinase 1

摘要

巨自噬/自噬将细胞质货物输送到溶酶体进行降解。在酵母中，单个 Atg8 蛋白在自噬体的形成中发挥作用，而在哺乳动物细胞中有五到七个旁系同源物，称为哺乳动物 Atg8s（mAtg8s：GABARAP、GABARAPL1、GABARAPL2、LC3A、LC3B、LC3B2 和 LC3C）与不完全定义的函数。在这里，我们表明 mAtg8s 的一个子集直接控制溶酶体生物发生。这发生在溶酶体转录程序的主要调节因子 TFEB 的水平上。mAtg8s 促进 TFEB 的核易位以响应饥饿等刺激。GABARAP 直接与 TFEB 相互作用，而 RNA-Seq 分析显示敲除六种编码 mAtg8s 的基因，或敲除编码所有 GABARAPs 的基因会减少 TFEB 转录程序。我们进一步表明，在饥饿期间需要 GABARAPs 与其他蛋白质、IRGM（一种与结核病和克罗恩病有关的因素）和 STX17 合作，以最佳地抑制 MTOR（TFEB 的上游激酶）和激活 PPP3/钙调磷酸酶磷酸酶。使 TFEB 去磷酸化，从而促进其核易位。总之，mAtg8s、IRGM 和 STX17 通过它们对 MTOR、TFEB 和 PPP3/钙调神经磷酸酶的组合或单独作用控制溶酶体生物发生，而与它们在自噬体膜形成中的作用无关。和激活使 TFEB 去磷酸化的 PPP3/钙调神经磷酸酶，从而促进其核易位。总之，mAtg8s、IRGM 和 STX17 通过它们对 MTOR、TFEB 和 PPP3/钙调神经磷酸酶的组合或单独作用控制溶酶体生物发生，而与它们在自噬体膜形成中的作用无关。和激活使 TFEB 去磷酸化的 PPP3/钙调神经磷酸酶，从而促进其核易位。总之，mAtg8s、IRGM 和 STX17 通过它们对 MTOR、TFEB 和 PPP3/钙调神经磷酸酶的组合或单独作用控制溶酶体生物发生，而与它们在自噬体膜形成中的作用无关。

缩写：AMPK：AMP活化蛋白激酶；IRGM：免疫相关的 GTPase M；mAtg8s：哺乳动物 Atg8 蛋白；MTOR：雷帕霉素激酶的机制靶点；PPP3CB：蛋白磷酸酶3催化亚基β；RRAGA：Ras 相关的 GTP 绑定 A.；STX17：syntaxin 17；ULK1：unc-51 像自噬激活激酶 1