PtdIns-specific MPR pathway association of a novel WD40 repeat protein, WIPI49.
WIPI49 is a member of a previously undescribed family of WD40-repeat proteins that we demonstrate binds 3-phosphorylated phosphoinositides. Immunofluorescent imaging indicates that WIPI49 is localized to both trans-Golgi and endosomal membranes, organelles between which it traffics in a microtubule-dependent manner. Live cell imaging establishes that WIPI49 traffics through the same set of endosomal membranes as that followed by the mannose-6-phosphate receptor (MPR), and consistent with this, WIPI49 is enriched in clathrin-coated vesicles. Ectopic expression of wild-type WIPI49 disrupts the proper functioning of this MPR pathway, whereas expression of a double point mutant (R221,222AWIPI49) unable to bind phosphoinositides does not disrupt this pathway. Finally, suppression of WIPI49 expression through RNAi, demonstrates that its presence is required for normal endosomal organization and distribution of the CI-MPR. We conclude that WIPI49 is a novel regulatory component of the endosomal and MPR pathway and that this role is dependent upon the PI-binding properties of its WD40 domain.
Jeffries
Tim R
TR
Protein Phosphorylation Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, United Kingdom.
Dove
Stephen K
SK
Michell
Robert H
RH
Parker
Peter J
PJ
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002402:Cathepsin D / Q000378:metabolism; D002522:Chlorocebus aethiops; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D003001:Cloning, Molecular; D011992:Endosomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D010802:Phylogeny; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D021601:trans-Golgi Network / Q000378:metabolism
D002352:Carrier Proteins; D010716:Phosphatidylinositols; D017527:Receptor, IGF Type 2; D002402:Cathepsin D