Negative regulation of autophagy by UBA6-BIRC6–mediated ubiquitination of LC3

Although the process of autophagy has been extensively studied, the mechanisms that regulate it remain insufficiently understood. To identify novel autophagy regulators, we performed a whole-genome CRISPR/Cas9 knockout screen in H4 human neuroglioma cells expressing endogenous LC3B tagged with a tandem of GFP and mCherry. Using this methodology, we identified the ubiquitin-activating enzyme UBA6 and the hybrid ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 as autophagy regulators. We found that these enzymes cooperate to monoubiquitinate LC3B, targeting it for proteasomal degradation. Knockout of UBA6 or BIRC6 increased autophagic flux under conditions of nutrient deprivation or protein synthesis inhibition. Moreover, UBA6 or BIRC6 depletion decreased the formation of aggresome-like induced structures in H4 cells, and α-synuclein aggregates in rat hippocampal neurons. These findings demonstrate that UBA6 and BIRC6 negatively regulate autophagy by limiting the availability of LC3B. Inhibition of UBA6/BIRC6 could be used to enhance autophagic clearance of protein aggregates in neurodegenerative disorders.