ABSTRACT

Depolarized mitochondria can be degraded via mitophagy, a selective form of autophagy. The RAB GTPase RAB7A was recently shown to play a key role in this process. RAB7A regulates late endocytic trafficking under normal growth conditions but is translocated to the mitochondrial surface following depolarization. However, how RAB7A activity is regulated during mitophagy is not understood. Here, using a proximity-dependent biotinylation approach (miniTurbo), we identified C5orf51 as a specific interactor of GDP-locked RAB7A. C5orf51 also interacts with the RAB7A guanine nucleotide exchange factor (GEF) complex members MON1 and CCZ1. In the absence of C5orf51, localization of RAB7A on depolarized mitochondria is compromised and the protein is degraded by the proteasome. Furthermore, depletion of C5orf51 also inhibited ATG9A recruitment to depolarized mitochondria. Together, these results indicate that C5orf51 is a positive regulator of RAB7A in its shuttling between late endosomes and mitochondria to enable mitophagy.

Abbreviations: ATG9A: autophagy related 9A; Baf A₁: bafilomycin A₁; BioID: proximity-dependent biotin identification; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CCZ1: CCZ1 homolog, vacuolar protein trafficking and biogenesis associated; DQ-BSA: dye quenched-bovine serum albumin; FYCO1: FYVE and coiled-coil domain autophagy adaptor 1; GAP: GTPase activating protein; GEF: guanine nucleotide exchange factor; KO: knockout; LRPPRC: leucine rich pentatricopeptide repeat containing; MG132: carbobenzoxy-Leu-Leu-leucinal; MON1: MON1 homolog, secretory trafficking associated; mtDNA: mitochondrial DNA; PINK1: PTEN induced kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RMC1: regulator of MON1-CCZ1; TBC1D15: TBC1 domain family member 15; TBC1D17: TBC1 domain family member 17; TOMM20: translocase of outer mitochondrial membrane 20; WDR91: WD repeat domain 91; WT: wild type.

摘要

去极化的线粒体可以通过线粒体自噬（一种选择性的自噬形式）降解。最近显示 RAB GTPase RAB7A 在此过程中发挥关键作用。RAB7A 在正常生长条件下调节晚期内吞转运，但在去极化后易位至线粒体表面。然而，在线粒体自噬过程中如何调节 RAB7A 活性尚不清楚。在这里，使用邻近依赖性生物素化方法 (miniTurbo)，我们将 C5orf51 鉴定为 GDP 锁定的 RAB7A 的特定相互作用物。C5orf51 还与 RAB7A 鸟嘌呤核苷酸交换因子 (GEF) 复合物成员 MON1 和 CCZ1 相互作用。在没有 C5orf51 的情况下，RAB7A 在去极化线粒体上的定位受到损害，蛋白质被蛋白酶体降解。此外，C5orf51 的消耗也抑制了 ATG9A 向去极化线粒体的募集。总之，这些结果表明 C5orf51 是 RAB7A 在晚期内体和线粒体之间穿梭以实现线粒体自噬的正调节因子。

缩写：ATG9A：自噬相关的9A；Baf A ₁ : 巴弗洛霉素 A ₁; BioID：邻近依赖的生物素识别；CCCP：羰基氰间氯苯腙；CCZ1：CCZ1 同源物，液泡蛋白运输和生物发生相关；DQ-BSA：染料淬灭-牛血清白蛋白；FYCO1：FYVE 和卷曲螺旋结构域自噬适配器 1；GAP：GTP酶激活蛋白；GEF：鸟嘌呤核苷酸交换因子；KO：淘汰赛；LRPPRC：富含亮氨酸的五肽重复序列；MG132：carbobenzoxy-Leu-Leu-leucinal；MON1：MON1 同源物，与分泌运输有关；mtDNA：线粒体DNA；PINK1：PTEN 诱导激酶 1；PRKN/PARKIN：parkin RBR E3泛素蛋白连接酶；RMC1：MON1-CCZ1的调节器；TBC1D15：TBC1 域家族成员 15；TBC1D17：TBC1 域家族成员 17；TOMM20：线粒体外膜转位酶 20；WDR91：WD 重复结构域 91；WT：野生型。