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Identification of EARP (Endosome-Associated Recycling Protein), a novel multisubunit tethering complex involved in endocytic recycling

Thursday, September 17, 2015 — Poster Session III

3:30 p.m. – 5:00 p.m.
FAES Terrace

* FARE Award Winner


  • Y Chen
  • C Schindler
  • J Pu
  • X Guo
  • JS Bonifacino


Material transfer between organelles of the endomembrane system is mediated by transport vesicles that bud from a donor compartment and fuse with an acceptor compartment. The first physical link between a transport vesicle and its acceptor compartment is established by tethering factors. Previous work showed that the multisubunit complex GARP (Golgi-Associated Retrograde Protein), composed of Ang2, Vps52, Vps53 and Vps54 subunits, functions as a tethering factor in retrograde transport from endosomes to the trans-Golgi network (TGN). In the current study, we found that a previously uncharacterized protein named “Syndetin” assembles with Ang2, Vps52 and Vps53 – but not Vps54 – to form another complex named “EARP” (Endosome-Associated Recycling Protein). Syndetin was identified as a top hit by mass spectrometry following tandem-affinity purification using Ang2, Vps52 and Vps53 as baits. Reciprocally, Ang2, Vps52 and Vps53 - but not Vps54 - were identified when Syndetin was used as the bait. Co-immunoprecipitation confirmed interactions of Syndetin with all the GARP subunits except for Vps54. In contrast to the TGN localization of Vps54 (i.e., GARP), co-localization analysis placed Syndetin (i.e., EARP) at Rab4-positive recycling endosomes. To establish the function of EARP, we depleted Syndetin in HeLa cells using siRNA and examined endocytic recycling and retrograde transport. Syndetin knockdown dramatically delayed the recycling of endocytosed transferrin receptor back to the cell surface, but had no effect on retrograde trafficking of fluorescently-labeled Shiga toxin B subunit to the Golgi complex. These studies thus identified EARP as a novel multisubunit tethering complex specifically involved in endocytic recycling.

Category: Cell Biology