Reconstitution of vesicle fusions occurring in endocytosis with a cell-free system.

摘要: We have used defined subcellular fractions to reconstitute in a cell-free system vesicle fusions occurring the endocytic pathway. The endosomal were prepared by immuno-isolation using as antigen an epitope located on foreign protein, transmembrane glycoprotein G (G-protein) of vesicular stomatitis virus. G-protein was first implanted cell plasma membrane and subsequently endocytosed for 15 30 min at 37 degrees C. immuno-isolated solid support cytoplasmic domain combination with specific monoclonal antibody. For comparative studies from cells absence internalization antibody against exoplasmic G-protein. vesicles contained 70% horseradish peroxidase internalized endosome fluid phase, exhibited acidic luminal pH shown acridine orange fluorescence differed their protein composition fraction. fusion originating different stages pathway studied assay both bio-chemical morphological detection system. These well provided recipient compartment (acceptor). They mixed post-nuclear supernatant containing endosomes loaded exogenous lactoperoxidase (donor) Fusion delivered donor lumen acceptor permitting fusion-specific iodination itself. required ATP detected only fraction after C but not or internalization.