Two distinct effectors of the small GTPase Rab5 cooperate in endocytic membrane fusion

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Abstract
Using the yeast two‐hybrid system, we have identified a novel 62 kDa coiled‐coil protein that specifically interacts with the GTP‐bound form of Rab5, a small GTPase that regulates membrane traffic in the early endocytic pathway. This protein shares 42% sequence identity with Rabaptin‐5, a previously identified effector of Rab5, and we therefore named it Rabaptin‐5β. Like Rabaptin‐5, Rabaptin‐5β displays heptad repeats characteristic of coiled‐coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP‐dependent manner. However, Rabaptin‐5β has features that distinguish it from Rabaptin‐5. The relative expression levels of the two proteins varies in different cell types. Rabaptin‐5β does not heterodimerize with Rabaptin‐5, and forms a distinct complex with Rabex‐5, the GDP/GTP exchange factor for Rab5. Immunodepletion of the Rabaptin‐5β complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin‐5 complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin‐5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin‐5 and Rabaptin‐5β complexes. Our results suggest that Rab5 binds to at least two distinct effectors which cooperate for optimal endocytic membrane docking and fusion.