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

Endosomes are a central sorting hub for membrane cargos. DNAJC13/RME-8 plays a critical role in endosomal trafficking by regulating the endosomal recycling or degradative pathways. DNAJC13 localizes to endosomes through its N-terminal Plekstrin Homology (PH)-like domain, which directly binds endosomal phosphoinositol-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-terminal tail, act as negative regulators of its PH-like domain. Using a structure-function approach combined with quantitative proteomics, we mapped these control points to a conserved YLT motif in the C-terminal tail as well as the catalytic HPD triad in its J domain. Mutation of either motif enhanced DNAJC13 endosomal localization in cells and increased binding to PI(3)P . Further, these effects required the N-terminal PH-like domain. We show that, similar to other PI(3)P binding domains, the N-terminal PH-like domain 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.