<scp>RNF13</scp> regulates the endolysosomal pathway through interaction with the small <scp>GTPase Arl8B</scp>

The endolysosomal system is a dynamic intracellular network essential for cargo degradation, recycling, and spatial compartmentalization. Proper coordination of endosome maturation and positioning is critical for lysosomal function and receptor fate. This study identifies an additional role for the E3 ubiquitin ligase RNF13 in controlling endolysosomal dynamics through its interaction with the small GTPase Arl8B. Predictive structural modeling and co‐immunoprecipitation revealed that RNF13 binds to Arl8B via residues Glu22 and Phe55 of Arl8B and Leu244 of RNF13. Binding occurs with a modest preference for the GDP‐bound Arl8B over GTP‐bound state, suggesting that RNF13 may engage an inactive fraction of Arl8B to influence endolysosomal positioning and assembly of Arl8B‐dependent trafficking complexes. Disrupting RNF13‐Arl8B binding alters Arl8B localization and redistributes lysosomes toward the cell periphery without changing the abundance of endolysosomal markers. Functionally, perturbing this interaction selectively alters epidermal growth factor receptor (EGFR) trafficking kinetics, consistent with a delayed progression of cargo toward lysosomal degradation rather than a general defect in endocytosis. Although overexpression of the Arl8B effector PLEKHM1 enhances RNF13‐Arl8B association, this is better explained by a shared Arl8B binding interface and does not imply direct cooperativity between RNF13 and PLEKHM1. Together, these findings identify RNF13 as a regulator of lysosomal organization and cargo transport, operating through Arl8B binding and ubiquitination that can occur without a proportional change in Arl8B abundance under our assay conditions. This work reveals an additional layer of regulation in endolysosomal trafficking, highlighting RNF13 as a regulatory node influencing cargo progression through degradative pathways.