Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes.
The molecular mechanisms ensuring directionality of endocytic membrane trafficking between transport vesicles and target organelles still remain poorly characterized. We have been investigating the function of the small GTPase Rab5 in early endocytic transport. In vitro studies have demonstrated a role of Rab5 in two membrane fusion events: the heterotypic fusion between plasma membrane-derived clathrin-coated vesicles (CCVs) and early endosomes and in the homotypic fusion between early endosomes. Several Rab5 effectors are required in homotypic endosome fusion, including EEA1, which mediates endosome membrane docking, as well as Rabaptin-5 x Rabex-5 complex and phosphatidylinositol 3-kinase hVPS34. In this study we have examined the localization and function of Rab5 and its effectors in heterotypic fusion in vitro. We report that the presence of active Rab5 is necessary on both CCVs and early endosomes for a heterotypic fusion event to occur. This process requires EEA1 in addition to the Rabaptin-5 complex. However, whereas Rab5 and Rabaptin-5 are symmetrically distributed between CCVs and early endosomes, EEA1 is recruited selectively onto the membrane of early endosomes. Our results suggest that EEA1 is a tethering molecule that provides directionality to vesicular transport from the plasma membrane to the early endosomes.
Rubino
M
M
European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Miaczynska
M
M
Lippé
R
R
Zerial
M
M
D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D002966:Clathrin; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D008565:Membrane Proteins; C097227:RABEP1 protein, human; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D020696:rab5 GTP-Binding Proteins