title: Reconstitution of endosomal proteolysis in a cell-free system. Transfer of immune complexes internalized via Fc receptors to an endosomal proteolytic compartment.
abstract: The presence of acid proteases in the endosomal compartment of macrophages has been recently demonstrated (Diment, S., Leech, M. S., and Stahl, P. D. (1988) J. Biol. Chem. 263, 6901-6907). This proteolytic activity allows the early degradation of ligands internalized by receptor-mediated endocytosis. To study the early steps that initiate the proteolytic processing of ligands, immune complexes formed with anti-dinitrophenol monoclonal IgG and radiolabeled dinitrophenol-derivatized bovine serum albumin were bound at 4 degrees C to Fc receptors of J774 macrophages. Cells were allowed to internalize immune complexes bound to the plasma membrane for different periods of time at 37 degrees C. Vesicle preparations generated from these cells were incubated in vitro at acidic pH to allow the hydrolysis of ligands located in protease-positive compartments. Ligand hydrolysis was observed after about 5 min of internalization, suggesting that at earlier times immune complexes were located in protease-free vesicles. Upon incubation of cell lysates under conditions that support in vitro endosome-endosome fusion, early protease-free endosomes containing ligand acquire proteolytic activity. Reconstitution of fusion-dependent proteolysis required energy, ions, membrane-associated factors, and cytosol. Cytosol was inactivated by incubation with N-ethylmaleimide. The proteolytic compartment formed upon in vitro incubation colocalized with endosomes in the light region of a Percoll gradient. Reconstitution was also achieved using an endosomal preparation separated from lysosomes in a Percoll gradient. Our results indicate that a fusion step between newly formed endocytic vesicles and a light density, protease-positive compartment triggers the proteolytic processing of ligands internalized by receptor-mediated endocytosis.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: L S
authors.initials: LS
authors.lastname: Mayorga
authors.forename: R
authors.initials: R
authors.lastname: Diaz
authors.forename: P D
authors.initials: PD
authors.lastname: Stahl
mesh_terms: D000818:Animals; D000936:Antigen-Antibody Complex; D002460:Cell Line; D002474:Cell-Free System; D004705:Endocytosis; D006868:Hydrolysis; D007700:Kinetics; D008264:Macrophages; D051379:Mice; D015388:Organelles / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011961:Receptors, Fc / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D000936:Antigen-Antibody Complex; D011961:Receptors, Fc; D010447:Peptide Hydrolases