Reconstitution of Rab- and SNARE-dependent membrane fusion by synthetic endosomes.
Rab GTPases and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are evolutionarily conserved essential components of the eukaryotic intracellular transport system. Although pairing of cognate SNAREs is sufficient to fuse membranes in vitro, a complete reconstitution of the Rab-SNARE machinery has never been achieved. Here we report the reconstitution of the early endosomal canine Rab5 GTPase, its key regulators and effectors together with SNAREs into proteoliposomes using a set of 17 recombinant human proteins. These vesicles behave like minimal 'synthetic' endosomes, fusing with purified early endosomes or with each other in vitro. Membrane fusion measured by content-mixing and morphological assays requires the cooperativity between Rab5 effectors and cognate SNAREs which, together, form a more efficient 'core machinery' than SNAREs alone. In reconstituting a fusion mechanism dependent on both a Rab GTPase and SNAREs, our work shows that the two machineries act coordinately to increase the specificity and efficiency of the membrane tethering and fusion process.
Ohya
Takeshi
T
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01309, Dresden, Germany.
Miaczynska
Marta
M
Coskun
Unal
U
Lommer
Barbara
B
Runge
Anja
A
Drechsel
David
D
Kalaidzidis
Yannis
Y
Zerial
Marino
M
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D011992:Endosomes / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008561:Membrane Fusion / Q000502:physiology*; D008854:Microscopy, Electron; D011510:Proteolipids / Q000378:metabolism / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D011510:Proteolipids; D011994:Recombinant Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; C020485:proteoliposomes; D020691:rab GTP-Binding Proteins