title: Receptor-mediated Endocytosis 8 Utilizes an N-terminal Phosphoinositide-binding Motif to Regulate Endosomal Clathrin Dynamics.
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.
authors.affiliation: From the Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
authors.forename: Besa
authors.initials: B
authors.lastname: Xhabija
authors.affiliation: From the Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada vacratsi@uwindsor.ca.
authors.forename: Panayiotis O
authors.initials: PO
authors.lastname: Vacratsis
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids; D002966:Clathrin / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D057809:HEK293 Cells; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D050827:Syntaxin 1 / Q000378:metabolism
chemical_list: D000596:Amino Acids; D002966:Clathrin; C507525:DNAJC13 protein, human; D050883:HSC70 Heat-Shock Proteins; D007295:Inositol Phosphates; D018832:Molecular Chaperones; D050505:Mutant Proteins; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D017527:Receptor, IGF Type 2; D050827:Syntaxin 1; C118500:inositol 3,4,5-trisphosphate; C106336:phosphatidylinositol 3,5-diphosphate; C055525:phosphatidylinositol 3-phosphate
keywords: 70-kilodalton heat shock protein (Hsp70); PI(3)P; RME-8; SNX1; clathrin; endocytosis; protein motif; protein sorting; protein-protein interaction; retrograde transport