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Reconstitution of vesicle fusions occurring in endocytosis with a cell‐free system.
Author(s) -
Gruenberg J.E.,
Howell K.E.
Publication year - 1986
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1986.tb04615.x
Subject(s) - biology , endocytosis , vesicle , microbiology and biotechnology , cell free system , cell , genetics , in vitro , membrane
We have used defined subcellular fractions to reconstitute in a cell‐free system vesicle fusions occurring in the endocytic pathway. The endosomal fractions were prepared by immuno‐isolation using as antigen an epitope located on a foreign protein, the transmembrane glycoprotein G (G‐protein) of vesicular stomatitis virus. The G‐protein was first implanted in the cell plasma membrane and subsequently endocytosed for 15 to 30 min at 37 degrees C. The endosomal fractions were immuno‐isolated on a solid support using as antigen the cytoplasmic domain of the G‐protein in combination with a specific monoclonal antibody. For comparative studies the plasma membrane was immuno‐isolated from cells in the absence of G internalization with a monoclonal antibody against the exoplasmic domain of the G‐protein. The immuno‐isolated endosomal vesicles contained 70% of horseradish peroxidase internalized in the endosome fluid phase, exhibited an acidic luminal pH as shown by acridine orange fluorescence and differed in their protein composition from the immuno‐isolated plasma membrane fraction. The fusion of endocytic vesicles originating from different stages of the pathway was studied in a cell‐free assay using both a bio‐chemical and a morphological detection system. These well defined endosomal vesicles were immuno‐isolated with the G‐protein on the solid support and provided the recipient compartment of the fusion (acceptor). They were mixed with a post‐nuclear supernatant containing endosomes loaded with exogenous lactoperoxidase (donor) at 37 degrees C. Fusion delivered the donor peroxidase to the lumen of acceptor vesicles permitting fusion‐specific iodination of the G‐protein itself. The fusion of vesicles required ATP and was detected only with an endosomal fraction prepared after internalization of the G‐protein for 15 min at 37 degrees C but not with a plasma membrane or with an endosomal fraction prepared after 30 min G‐protein internalization.