GTPase activity of Rab5 acts as a timer for endocytic membrane fusion.
The GTPase cycle is a versatile regulatory mechanism directing many cell functions, and Rab family members use it to regulate intracellular transport. Current models propose that GTP hydrolysis by Rab proteins is either required for membrane fusion or occurs afterwards to allow recycling of the protein. To measure the GTPase activity of Rab5 in endocytic membrane fusion, we engineered a mutant that preferentially binds xanthosine 5'-triphosphate (XTP),Rab5(D136N) and monitored the kinetics of [alpha(32)P]-XTP hydrolysis in situ during endosome fusion in vitro. Surprisingly, nucleotide hydrolysis occurred even in the absence of membrane fusion, indicating that membrane-bound Rab5 undergoes futile cycles of GTP(XTP) binding and hydrolysis. Nucleotide triphosphate hydrolysis by Rab5 is not conditional on membrane fusion and is reduced by its effector Rabaptin-5. Our data reveal that the GTP cycle of Rab proteins differs from that of other GTPases (for example, EF-Tu) and indicate that GTP hydrolysis acts as a timer that determines the frequency of membrane docking/fusion events.
Rybin
V
V
European Molecular Biology Laboratory, Heidelberg, Germany.
Ullrich
O
O
Rubino
M
M
Alexandrov
K
K
Simon
I
I
Seabra
M C
MC
Goody
R
R
Zerial
M
M
D020727:ADP-Ribosylation Factors; D002474:Cell-Free System; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000378:metabolism / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins
D008565:Membrane Proteins; D011994:Recombinant Proteins; D012265:Ribonucleotides; D033921:Vesicular Transport Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C065326:xanthosine 5'-triphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D020696:rab5 GTP-Binding Proteins