巨自噬,下文简称为自噬,是在真核生物中高度保守的细胞内降解系统。由于自噬过程伴随着膜动力学,RAB小GTPases,膜贩运的关键监管机构,通常认为调节自噬的膜动力学。尽管据报道,超过一半的哺乳动物RAB参与了规范和选择性的自噬,关于RAB在哺乳动物自噬中的作用尚未达成共识.这里,我们全面分析了MDCK细胞的rab敲除(KO)文库,以重新评估基础和饥饿诱导的自噬中每个RAB亚型的需求.结果显示,仅在四个rab-KO细胞(rab1-KO,rab2-KO,rab7a-KO,和rab14-KO细胞),并将RAB14鉴定为自噬的新调节剂,特别是在自噬体成熟步骤。其中两个rab-KO细胞的自噬缺陷表型,rab2-KO和rab14-KO细胞,非常温和,但是rab2和rab14的双重KO导致了更严重的自噬缺陷表型(LC3积累大于单个KO细胞,表明RAB2和RAB14在自噬成熟过程中的重叠作用。我们还发现RAB14在系统发育上与RAB2相似,并且具有与RAB2相同的特性,即自噬体定位以及与HOPS亚基VPS39和VPS41的相互作用。我们的发现表明,RAB2和RAB14通过将HOPS复合物募集到自噬体来重叠调节自噬体成熟步骤。
Macroautophagy, simply referred to below as autophagy, is an intracellular degradation system that is highly conserved in eukaryotes. Since the processes involved in autophagy are accompanied by membrane dynamics, RAB small GTPases, key regulators of membrane trafficking, are generally thought to regulate the membrane dynamics of autophagy. Although more than half of the mammalian RABs have been reported to be involved in canonical and selective autophagy, no consensus has been reached in regard to the role of RABs in mammalian autophagy. Here, we comprehensively analyzed a rab-knockout (KO) library of MDCK cells to reevaluate the requirement for each RAB isoform in basal and starvation-induced autophagy. The results revealed clear alteration of the MAP1LC3/LC3-II level in only four rab-KO cells (rab1-KO, rab2-KO, rab7a-KO, and rab14-KO cells) and identified RAB14 as a new regulator of autophagy, specifically at the autophagosome maturation step. The autophagy-defective phenotype of two of these rab-KO cells, rab2-KO and rab14-KO cells, was very mild, but double KO of rab2 and rab14 caused a severer autophagy-defective phenotype (greater LC3 accumulation than in single-KO cells, indicating an overlapping role of RAB2 and RAB14 during autophagosome maturation. We also found that RAB14 is phylogenetically similar to RAB2 and that it possesses the same properties as RAB2, i.e. autophagosome localization and interaction with the HOPS subunits VPS39 and VPS41. Our findings suggest that RAB2 and RAB14 overlappingly regulate the autophagosome maturation step through recruitment of the HOPS complex to the autophagosome.Abbreviation: AID2: auxin-inducible degron 2; ATG: autophagy related; BafA1: bafilomycin A1; CKO: conditional knockout; EBSS: Earle\'s balanced salt solution; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; HRP: horseradish peroxidase; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; MDCK: Madin-Darby canine kidney; mAb: monoclonal antibody; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; 5-Ph-IAA: 5-phenyl-indole-3-acetic acid; pAb: polyclonal antibody; siRNA: small interfering RNA; SNARE: soluble NSF-attachment protein receptor; TF: transferrin; WT: wild-type.