Inhibition of endocytic transport by aluminum fluoride implicates GTPases as regulators of endocytosis.
It is now well established that GTP-binding proteins are important regulators of vesicular transport. Recent work has shown that multiple GTPases (both monomeric and heterotrimeric) are required for trafficking. In the present study we have used aluminum fluoride (AIF), a reagent that activates trimeric G proteins, as a tool to study the involvement of this family of GTPases in the regulation of endocytosis in intact cells. Our results indicate that AIF inhibits fusion of early endosomes with an intracellular proteolytic compartment. Using the mixing of sequentially internalized ligands as a measure of endocytosis, we found that AIF inhibited endocytic transport as assessed by both biochemical and morphological methods. Taken together these results suggest that AIF affects membrane fusion, a common step in vesicular transport. To further examine the effects of AIF we tested this compound in a cell-free assay that reconstitutes fusion among endosomes. AIF affected endosomal fusion in a different way than did GTP gamma S, an agent that activates both trimeric and small GTPases. Our results suggest that the coordinated activation of both classes of GTPases are required for efficient endocytic transport.
Colombo
M I
MI
Department of Cell Biology and Physiology, Washington University, School of Medicine, St Louis 63110.
Lenhard
J
J
Mayorga
L
L
Beron
W
W
Hall
H
H
Stahl
P D
PD
D017607:Aluminum Compounds / Q000494:pharmacology*; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000502:physiology; D004705:Endocytosis / Q000187:drug effects* / Q000502:physiology*; D011992:Endosomes / Q000187:drug effects / Q000502:physiology; D005459:Fluorides / Q000494:pharmacology*; D020558:GTP Phosphohydrolases / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology
D017607:Aluminum Compounds; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020558:GTP Phosphohydrolases; D005459:Fluorides; C032311:aluminum fluoride