Abstract
Receptor-mediated endocytosis 8 (RME-8) is a DnaJ domain containing protein implicated in translocation of Hsc70 to early endosomes for clathrin removal during retrograde transport. Previously, we have demonstrated that RME-8 associates with early endosomes in a phosphatidylinositol 3-phosphate (PI(3)P)-dependent fashion. In this study, we have now identified amino acid determinants required for PI(3)P binding within a region predicted to adopt a pleckstrin homology-like fold in the N terminus of RME-8. The ability of RME-8 to associate with PI(3)P and early endosomes is largely abolished when residues Lys(17), Trp(20), Tyr(24), or Arg(26) are mutated resulting in diffuse cytoplasmic localization of RME-8 while maintaining the ability to interact with Hsc70. We also provide evidence that RME-8 PI(3)P binding regulates early endosomal clathrin dynamics and alters the steady state localization of the cation-independent mannose 6-phosphate receptor. Interestingly, RME-8 endosomal association is also regulated by the PI(3)P-binding protein SNX1, a member of the retromer complex. Wild type SNX1 restores endosomal localization of RME-8 W20A, whereas a SNX1 variant deficient in PI(3)P binding disrupts endosomal localization of wild type RME-8. These results further highlight the critical role for PI(3)P in the RME-8-mediated organizational control of various endosomal activities, including retrograde transport.
Highlights
Receptor-mediated endocytosis 8 (RME-8) is involved in clathrin removal at early endosomes
Identification of the RME-8 PI[3]P-binding Motif—Earlier studies have shown that RME-8 associates with early endosomes [6, 7, 18], and recently our laboratory has discovered RME-8 to be a novel PI[3]P-binding protein that is regulated by the PI[3]P phosphatase MTMR2 [24]
The primary sequence of RME-8 was analyzed by the Protein Homology/AnalogY Recognition Engine Version 2.0 (Phyre2) protein folding prediction program [26, 27]
Summary
RME-8 is involved in clathrin removal at early endosomes. Results: RME-8 possesses a novel PI[3]P-binding motif within its N terminus. Receptor-mediated endocytosis 8 (RME-8) is a DnaJ domain containing protein implicated in translocation of Hsc70 to early endosomes for clathrin removal during retrograde transport.
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