In vitro reconstitution of phagosome-endosome fusion: evidence for regulation by heterotrimeric GTPases.
We have assessed the role of heterotrimeric GTPases on in vitro fusion of phagosomes and endosomes. Highly purified phagosomes were found to contain G alpha s, G alpha i1, G alpha i2, G alpha i3, and G beta subunits of heterotrimeric GTP-binding proteins. A functional role for G proteins was established using an in vitro phagosome-endosome fusion assay. First, addition of AlF4- and purified G beta gamma subunits to the in vitro assay blocked fusion, indicating that heterotrimeric G proteins may play a role, either direct or indirect, in phagosome maturation. Second, a striking inhibitory effect was observed when the vesicles were incubated with peptides that preferentially activate G alpha s. A similar effect on phagosome-endosome fusion was observed with cholera toxin, a reagent known to activate G alpha s. Our results suggest that one or more heterotrimeric G proteins, including Gs, mediate and/or regulate phagosome-endosome fusion.
Berón
W
W
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Colombo
M I
MI
Mayorga
L S
LS
Stahl
P D
PD
D017607:Aluminum Compounds / Q000494:pharmacology; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D011992:Endosomes / Q000502:physiology* / Q000648:ultrastructure; D005459:Fluorides / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007425:Intracellular Membranes / Q000737:chemistry / Q000502:physiology; D046911:Macromolecular Substances; D008561:Membrane Fusion / Q000187:drug effects; D009243:NAD / Q000494:pharmacology; D010588:Phagosomes / Q000502:physiology* / Q000648:ultrastructure; D011189:Potassium Chloride / Q000494:pharmacology
D017607:Aluminum Compounds; D046911:Macromolecular Substances; D009243:NAD; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011189:Potassium Chloride; D002772:Cholera Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D005459:Fluorides; C032311:aluminum fluoride