RNF13 Regulates the Endolysosomal Pathway Through Interaction with the Small GTPase Arl8B

The endolysosomal system is a dynamic intracellular network essential for cargo degradation, recycling and spatial compartmentalization. Precise coordination of endosome maturation and positioning is critical for maintaining lysosomal function and regulating receptor fate. This study uncovers a novel role for the E3 ubiquitin ligase RNF13 in controlling endolysosomal dynamics through its interaction with the small GTPase Arl8B. Using predictive structural modeling and co-immunoprecipitation assays, the results demonstrate that RNF13 binds to Arl8B, implicating residues Glu22 and Phe55 of Arl8B with RNF13s Leu244. Their interaction influences lysosomal positioning and the trafficking of endocytic cargo. Notably, loss of RNF13-Arl8B binding alters Arl8B localization and causes a peripheral redistribution of lysosomes, while not affecting the abundance of endolysosomal markers. However, it does impair the internalization of the epidermal growth factor receptor (EGFR). These findings suggest that the RNF13-Arl8B interaction plays a crucial role in modulating vesicle maturation and fusion. Furthermore, overexpression of the Arl8B effector PLEKHM1 enhances RNF13-Arl8B complex formation, indicating a possible cooperative assembly of tethering complexes during lysosome- endosome fusion. Together, the results identify RNF13 as a spatial regulator of lysosomal organization and cargo processing, operating through a non-enzymatic scaffolding mechanism. This reveals an additional layer of regulation in endolysosomal trafficking, supporting a role for RNF13 as a checkpoint in cargo progression through degradative pathways. Altogether, the results of this work expand the understanding of the molecular coordination underlying lysosomal dynamics and underscore the importance of selective effector interactions in coordinating endolysosomal trafficking.