Isolation of functional, coated, endocytic vesicles.
Brief internalization of [125I]transferrin was used to label coated endocytic vesicles, which were then purified using a combination of 2H2O and 2H2O/Ficoll density gradients. Purification was monitored using an assay measuring fusion of endocytic organelles, so as to isolate functional vesicles. Isolated vesicles had all the properties of clathrin-coated vesicles, being enriched for the major components of clathrin coats and uncoated by either 1 M Tris-HCl or an uncoating ATPase. Nearly half of the labeled vesicles were able to participate in subsequent fusion events, as measured by the cell-free assay. Fusion was specific, requiring energy and cytosol, and being sensitive to N-ethyl maleimide.
Woodman
P G
PG
Cell Biology Laboratory, Imperial Cancer Research Fund, London, England.
Warren
G
G
D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods; D002966:Clathrin / Q000302:isolation & purification / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000648:ultrastructure*; D003676:Deferoxamine / Q000494:pharmacology; D004705:Endocytosis; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D008715:Methionine / Q000378:metabolism; D008854:Microscopy, Electron; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure*; D014168:Transferrin / Q000378:metabolism*
D002966:Clathrin; D008565:Membrane Proteins; D014168:Transferrin; D008715:Methionine; D003676:Deferoxamine