Endosomal density shift is related to a decrease in fusion capacity.
Dinitrophenol (DNP)-beta-glucuronidase and mannosylated anti-DNP IgG, which are endocytosed by the mannose receptor and delivered to lysosomes, were previously developed as probes for examination of fusion between early endosomes in a cell-free system. In this study, these probes were found to be transported by intact cells to endocytic vesicles with heavy buoyant density at different rates, as determined by Percoll gradient fractionation of cell homogenates. There was a concomitant loss of in vitro fusion activity as the ligands moved to dense compartments. In monensin-treated cells, DNP-beta-glucuronidase was retained in a light compartment corresponding to intracellular vesicles capable of fusion in vitro. Pulse-chase studies using a DNP-derivatized transferrin-alkaline phosphatase conjugate showed that a recycling ligand was always found in light intracellular vesicles that were capable of fusion to early endosomes in vitro. In contrast to cell-free systems, intact cells sequentially labeled with DNP-beta-glucuronidase and then mannosylated anti-DNP IgG showed ligand mixing in both early and late endocytic compartments. Treatment with nocodazole or colchicine did not affect the rate of DNP-beta-glucuronidase transport to heavy vesicles in intact cells, however, the extent of ligand mixing in late endosomes was decreased by microtubule disruption. Using sequentially labeled cells split into two groups, we directly compared ligand mixing in vitro to mixing by intact cells. Fusion alone does not mediate increases in vesicle density, since DNP-beta-glucuronidase/anti-DNP IgG complexes formed in vitro were found in light vesicles, while intact cells showed immune complexes predominantly in heavy vesicles. These results suggest that the density shift is an initial step in targeting to lysosomes.
Diaz
R
R
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis 63110.
Colombo
M I
MI
Koval
M
M
Mayorga
L
L
Stahl
P
P
D000818:Animals; D001693:Biological Transport, Active; D015415:Biomarkers; D002499:Centrifugation, Density Gradient; D002999:Clone Cells; D004140:Dinitrophenols / Q000276:immunology / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D005966:Glucuronidase / Q000276:immunology / Q000378:metabolism; D008264:Macrophages / Q000502:physiology* / Q000648:ultrastructure; D008561:Membrane Fusion / Q000502:physiology*; D014617:Vacuoles / Q000502:physiology* / Q000648:ultrastructure
D015415:Biomarkers; D004140:Dinitrophenols; D005966:Glucuronidase