DNAJC13 localization to endosomes is opposed by its J domain and its disordered C-terminus.

Endosomes are a central sorting hub for membrane cargos. DNAJC13/RME-8 plays a critical role in endosomal trafficking by regulating the endosomal recycling and degradative pathways. DNAJC13 localizes to endosomes through its N-terminal Pleckstrin Homology (PH)-like domain, which binds endosomal phosphatidylinositol-3-phosphate (PI(3)P). However, little is known about how DNAJC13 localization is regulated. Here, we show that two regions within DNAJC13, its J domain and disordered C-terminus, act as negative regulators of its PH-like domain. Using a structure-function approach, we map these control points to a conserved YLT motif in the disordered C-terminus as well as the catalytic HPD triad in its J domain. Mutation of either motif enhances DNAJC13 endosomal localization in cells and increases binding to PI(3)P in vitro, and overexpression of these mutants cause multiple defects in endosome function, including endosomal clustering and loss of recycling of a membrane protein cargo. Mechanistically, the enhanced endosomal localization of DNAJC13 requires its N-terminal PH-like domain, and we show that the PH-like domain of DNAJC13 binds PI(3)P weakly in isolation and requires oligomerization for efficient PI(3)P binding and endosomal localization. Together, these results demonstrate that interaction between DNAJC13 and PI(3)P serves as a molecular control point for regulating DNAJC13 localization to endosomes.