Two 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 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-5beta. Like Rabaptin-5, Rabaptin-5beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner. However, Rabaptin-5beta has features that distinguish it from Rabaptin-5. The relative expression levels of the two proteins varies in different cell types. Rabaptin-5beta 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-5beta 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-5beta complexes. Our results suggest that Rab5 binds to at least two distinct effectors which cooperate for optimal endocytic membrane docking and fusion.
Gournier
H
H
European Molecular Biology Laboratory, Postfach 10.2209, D-69012 Heidelberg, Germany.
Stenmark
H
H
Rybin
V
V
Lippé
R
R
Zerial
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D003001:Cloning, Molecular / Q000379:methods; D003600:Cytosol / Q000737:chemistry; D019281:Dimerization; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006367:HeLa Cells; D006801:Humans; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D033921:Vesicular Transport Proteins; D015003:Yeasts / Q000235:genetics; D020696:rab5 GTP-Binding Proteins
D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; C097227:RABEP1 protein, human; C112197:RABEP2 protein, human; C108382:RABGEF1 protein, human; C486276:Rabep1 protein, mouse; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins