The Rab5 effector EEA1 is a core component of endosome docking

Intracellular membrane docking and fusion requires the interplay between so luble factors and SNAREs. The SNARE hypothesis' postulates that pairing bet ween a vesicular v-SNARE and a target membrane z-SNARE is the primary molec ular interaction underlying the specificity of vesicle targeting as well as lipid bilayer fusion. This proposal is supported by recent studies using a minimal artificial system(2). However, several observations demonstrate th at SNAREs function at multiple transport steps and can pair promiscuously, questioning the role of SNAREs in conveying vesicle targeting(3-6). Moreove r, other proteins have been shown to be important in membrane docking or te thering(7-9). Therefore, if the minimal machinery is defined as the set of proteins sufficient to reproduce in vitro the fidelity of vesicle targeting , docking and fusion as in vivo, then SNAREs are not sufficient to specify vesicle targeting. Endosome fusion also requires cytosolic factors and is r egulated by the small GTPase Rab5 (refs 10-20). Here we show that Rab5-inte racting soluble proteins can completely substitute for cytosol in an in viv o endosome-fusion assay, and that the Rab5 effector EEA1 is the only factor necessary to confer minimal fusion activity. Rab5 and other associated pro teins seem to act upstream of EEA1, implying that Ra65 effecters comprise b oth regulatory molecules and mechanical components of the membrane transpor t machinery. We further show that EEA1 mediates endosome docking and, toget her with SNAREs, leads to membrane fusion.