Relationships between EEA1 binding partners and their role in endosome fusion

Homotypic fusion between early endosomes requires the phosphatidylinositol 3-phosphate (PI3P)-binding protein, Early Endosomal Autoantigen 1 (EEA1), W e have investigated the role of other proteins that interact with EEA1 in t he fusion of early endosomes derived from Baby Hamster Kidney (BHK) cells, We confirm a requirement for syntaxin 13, but additionally show that the as say is equally sensitive to reagents specifically targeted against syntaxin 6, Binding of EEA1 to immobilised GST-syntaxin 6 and 13 was directly compa red; only syntaxin 6 formed a stable complex with EEA1, Early endosome fusi on requires the release of intravesicular calcium, and calmodulin plays a v ital role in the fusion pathway, as judged by sensitivity to antagonists. W e demonstrate that both EEA1 and syntaxin 13 interact with calmodulin, In t he case of EEA1, binding to calmodulin requires an IQ domain, which is adja cent to a C-terminal FYVE domain that specifically binds to PI3P, We have a ssessed the influence of protein binding partners on EEA1 interaction with PI3P and find that both calmodulin and rab5-GTP are antagonistic to PI3P bi nding, whilst syntaxins 6 and 13 have no effect. These studies reveal a com plex network of interactions between the proteins required for endosome fus ion.