Characterization of Rab5:Q79L-stimulated endosome fusion.
Fusion of intracellular membrane-bound compartments is a common step in the transport of macromolecules along the endocytic and secretory pathways. Previous work has shown that GTP gamma S stimulates endosome fusion in the presence of low concentrations of cytosol. In this study, we have characterized the effect of rab5:Q79L, a mutant with reduced GTPase activity, on endosome fusion in a cell-free assay. rab5:Q79L stimulates in vitro endosome fusion. The stimulatory effects required ATP, were blocked by N-ethylmaleimide (NEM) and anti-NEM-sensitive fusion (NSF) protein antibody, but could proceed in the absence of cytosol. Stimulation of fusion with rab5:Q79L led to rapid inactivation of the vesicles when tested in a second incubation for fusogenic activity. By electron microscopy, endosomes connected by tubular structures were frequently observed in the presence of rab5:Q79L. Rab5:Q79L promoted fusion only among early endosomes; when the ligands were chased into more mature endocytic compartments, fusion was not observed. Phospholipase A2 inhibitors blocked rab5:Q79L-stimulated fusion. The results indicate that rab5:Q79L promotes fusion by activating factors already present in the membranes and that NSF and phospholipase A2 activities are required downstream of rab5.
Barbieri
M A
MA
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Li
G
G
Mayorga
L S
LS
Stahl
P D
PD
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002474:Cell-Free System; D011992:Endosomes / Q000648:ultrastructure*; D020558:GTP Phosphohydrolases / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008561:Membrane Fusion / Q000187:drug effects*; D051379:Mice; D008854:Microscopy, Electron; D009154:Mutation; D052066:N-Ethylmaleimide-Sensitive Proteins; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins
D002352:Carrier Proteins; D033921:Vesicular Transport Proteins; D000255:Adenosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; C498733:Nsf protein, mouse; D020696:rab5 GTP-Binding Proteins