The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts.

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Abstract
Recent work from several laboratories suggested the participation of intermediate structures in the delivery of adsorbed ligands from the plasma membrane to lysosomes. Subcellular fractionation studies are presented bearing on the role of these structures in adsorptive pinocytosis of epidermal growth factor (EGF), .beta.-hexosaminidase and low density lipoprotein (LDL) by human fibroblasts. A 2-step Percoll density gradient fractionation, identified newly internalized (5 min) EGF in 2 intermediate density structures that are essentially negative for plasma membrane marker, and more bouyant than secondary lysosomes. Continued incubation for 20 min resulted in transfer to (or conversion to) vesicles sedimenting with secondary lysosomes. Internalized .beta.-hexosaminidase and LDL behaved similarly, appearing first in structures of intermediate density and later appearing in association with secondary lysosomes. Two drugs, NH4Cl and monensin, were found to inhibit ligand transfer to the secondary lysosome peak, although they did not inhibit entry of bound ligands into intermediate density structures. Upon removal of both inhibitors, internalized ligands were quickly transferred to the secondary lysosome peak. This transfer process was faster for EGF than for the other 2 ligands studied. The endocytosis of these 3 ligands, and their delivery to lysosomes in fibroblasts, evidently proceeds through a common pathway involving intermediate nonlysosomal structures.