Bouchard, A. Y. | Search bioRxiv

bioRxiv2025

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.

cell biology↗

bioRxiv2025

RNF13 is a novel interactor of iduronate 2-sulfatase that modifies its glycosylation and maturation

Mucopolysaccharidosis type II, also known as Hunter syndrome, is a rare and fatal disease caused by mutations in the iduronate 2-sulfatase (IDS) encoding gene. The enzymatically inactive variant proteins lead to pathological accumulation of glycosaminoglycans in the lysosomes, causing dysfunction in multiple organs. IDS is expressed as a precursor protein, and its processing and lysosomal targeting are crucial for proper enzymatic activity. However, IDS intracellular dynamic is poorly understood and a better understanding of its processing mechanisms would benefit the development of new therapeutic strategies. AlphaFold 3 predicted an interaction between IDS and the E3 ubiquitin ligase RNF13. Co-immunoprecipitation assays confirm this interaction and further show that RNF13 interacts preferentially with a predominantly underglycosylated immature form of IDS, resulting in altered IDS glycosylation and maturation. The results demonstrate that IDS glycosylation site Asn246 is important for lysosomal targeting, although its glycosylation is not altered by RNF13. This study also unravels that RNF13 forms a heterodimer with the E3 ubiquitin ligase RNF167 that modify both RNF13 and RNF167 lysosomal trafficking. In addition, the heterodimer interacts and alters IDS differently than RNF13 or RNF167 alone. RNF13 catalytic E3 ligase activity is required to generate an underglycosylated form, but not that of RNF167. This study exposes that the proteasome rapidly degrades IDS underglycosylated forms, and RNF13 exerts a protective effect. Overall, this study reveals a novel and dual role of RNF13 on IDS maturation and degradation, providing mechanistic insights into IDS trafficking.

biochemistry↗