Comprehensive knockout analysis of the RAB family small GTPases reveals an overlapping role of RAB2 and RAB14 in autophagosome maturation.

Abstract

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.