2 DISTINCT EFFECTORS OF THE SMALL GTPASE RAB5 COOPERATE IN ENDOCYTIC MEMBRANE-FUSION

Using the yeast two-hybrid system, we have identified a novel 62 kDa c oiled-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 Raba ptin-5, a previously identified effector of Rab5, and we therefore nam ed it Rabaptin-5 beta. Like Rabaptin-5, Rabaptin-5 beta displays hepta d repeats characteristic of coiled-coil proteins and is recruited on t he endosomal membrane by Rab5 in a GTP-dependent manner. However, Raba ptin-5 beta has features that distinguish it from Rabaptin-5. The rela tive expression levels of the two proteins varies in different cell ty pes. Rabaptin-5 beta does not heterodimerize with Rabaptin-5, and form s a distinct complex with Rabex-5, the GDP/GTP exchange factor for Rab 5. Immunodepletion of the Rabaptin-5 beta complex from cytosol only pa rtially inhibits early endosome fusion in vitro, whereas the additiona l 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 o f both Rabaptin-5 and Rabaptin-5 beta complexes. Our results suggest t hat Rab5 binds to at least two distinct effecters which cooperate for optimal endocytic membrane docking and fusion.