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Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues
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PtdIns-specific MPR pathway association of a novel WD40 repeat protein, WIPI49

Tim R Jeffries1, Stephen K Dove, Robert H Michell

  • 1Protein Phosphorylation Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, United Kingdom.

Molecular Biology of the Cell
|March 17, 2004
PubMed

Insights

WIPI49, a novel WD40-repeat protein, binds phosphoinositides and regulates the endosomal and mannose-6-phosphate receptor (MPR) pathway. Its function depends on PI-binding, impacting endosomal organization and MPR distribution.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • WD40-repeat proteins are involved in diverse cellular functions.
  • Phosphoinositides are key signaling lipids regulating membrane trafficking.
  • The mannose-6-phosphate receptor (MPR) pathway is crucial for lysosomal enzyme sorting.

Purpose of the Study:

  • To characterize a novel WD40-repeat protein, WIPI49.
  • To investigate WIPI49's role in phosphoinositide binding and membrane trafficking.
  • To determine WIPI49's involvement in the MPR pathway.

Main Methods:

  • Immunofluorescent imaging to determine WIPI49 localization.
  • Live cell imaging to track WIPI49 trafficking.
  • Analysis of MPR pathway function in cells expressing wild-type or mutant WIPI49.
  • RNA interference (RNAi) to suppress WIPI49 expression.

Main Results:

  • WIPI49 binds 3-phosphorylated phosphoinositides.
  • WIPI49 localizes to trans-Golgi and endosomal membranes and traffics in a microtubule-dependent manner.
  • WIPI49 shares trafficking pathways with MPR and is enriched in clathrin-coated vesicles.
  • Ectopic expression of wild-type WIPI49 disrupts the MPR pathway, while a PI-binding mutant does not.
  • WIPI49 suppression affects endosomal organization and CI-MPR distribution.

Conclusions:

  • WIPI49 is a novel regulatory component of the endosomal and MPR pathway.
  • WIPI49's role is dependent on its phosphoinositide-binding properties mediated by its WD40 domain.
  • WIPI49 is essential for normal endosomal organization and CI-MPR trafficking.

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