Abstract
Autophagy is a tightly regulated process essential for cellular homeostasis, with ubiquitination playing a crucial role in its regulation. However, the specific ubiquitin related factors involved in autophagic flux remain largely unexplored. Identifying these regulators is essential for advancing the mechanistic understanding of autophagy and its broader implications in cellular function. This study aimed to identify novel ubiquitination-associated regulators of autophagy. To achieve this, we conducted a CRISPR-Cas9 loss-of-function screen targeting 660 ubiquitination-related genes in pancreatic cancer cells expressing the mCherry-GFP-LC3 autophagy flux reporter system. Among the top candidates, we identified G2E3, a G2/M-phase-specific E3 ubiquitin ligase, as a previously unrecognized autophagy regulator. Subsequent functional analyses revealed that G2E3 knock out led to a significant accumulation of LC3B-II and GABARAPs, indicative of impaired autophagic flux. Further confocal imaging demonstrated that the co-localization of LC3B with LAMP1-positive lysosomes was significantly reduced in G2E3 knock out cells, suggesting defective autophagosome-lysosome fusion. Mechanistically, G2E3 directly interacts with GABARAP and GABARAPL1, but not LC3B, positioning it as a key regulator of late-stage autophagy. Additionally, G2E3 knock out cells exhibited reduction in migration and invasion capability, suggesting its role in cancer progression. These findings establish G2E3 as a novel ubiquitin-related regulator of autophagy, specifically facilitating autophagosome-lysosome fusion via a GABARAPs-dependent mechanism. This study reveals a previously unrecognized role of G2E3 in late-stage autophagy and suggests that targeting G2E3 could provide a potential therapeutic approach for modulating autophagy-dependent cellular processes, including cancer progression.