High-yield isolation of functionally competent endosomes from mouse lymphocytes.
A discontinuous-sucrose-gradient procedure for isolating endosomes from mouse lymphoma cells has been developed. After centrifugation, most organelles (especially mitochondria and lysosomes) are recovered in the denser fractions of the gradient, whereas a mixture of plasma membrane and endosomes is present at lighter densities. The endosome recovery in this fraction can be increased (by 100%) by (a) a mild trypsin treatment of the postnuclear supernatant and (b) loading the cell endosomes with a saturating concentration of low-density lipoproteins. Removal of the plasma-membrane contamination was achieved by preincubating the cells with a gold-ricin complex at 4 degrees C. On centrifugation, the gold-loaded membranes sediment to the bottom of the gradient. The endosome preparation isolated by these procedures is less than 6% contaminated by other organelles and contains 42% of internalized 125I-transferrin. We show that these isolated endosomes are functional, as displayed by their ability to fuse and to acidify in a cell-free system. Endosome fusion was studied by a new assay based on the use of fluorescence resonance energy transfer. This fusion is dependent on ATP and on a cytosolic, thermoresistant but trypsin- and N-ethylmaleimide-sensitive, protein factor. Early endosomes fuse more actively among themselves than with late-endocytic vesicles, and they fuse only slowly with plasma-membrane vesicles.
Beaumelle
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BD
Department of Biochemistry, Imperial College of Science and Technology, London, U.K.
Hopkins
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CR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002451:Cell Compartmentation; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000502:physiology; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D008077:Lipoproteins, LDL / Q000378:metabolism; D008214:Lymphocytes / Q000648:ultrastructure*; D008561:Membrane Fusion; D051379:Mice; D008854:Microscopy, Electron; D015388:Organelles; D012276:Ricin / Q000008:administration & dosage; D014168:Transferrin / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D002413:Cations, Divalent; D008077:Lipoproteins, LDL; D014168:Transferrin; D000255:Adenosine Triphosphate; D012276:Ricin; D014357:Trypsin