Separation of Two Populations of Endocytic Vesicles Involved in Receptor-ligand Sorting in Rat Hepatocytes

Rat hepatocytes incubated in high K+ buffer (all Na+ is replaced by K+) internalize glycoproteins bearing terminal galactose moieties but are not able to deliver them to lysosomes (Baenziger, J. U., and Fiete, D. (1982) J. Biol. Chem. 257, 6007-6009). Instead, internalized ligand accumulates in a prelysosomal compartment(s) with a density similar to that of plasma membrane. We have separated two populations of prelysosomal endocytic vesicles from hepatocytes incubated in high K+ buffer. The vesicle population VR.L has a mean density of 1.14 by sucrose gradient centrifugation and contains functionally active Gal/GalNAc-specific receptor which is able to bind intravesicular ligand. The vesicle population VL has a mean density of 1.19. It contains ligand, but is deficient in Gal/GalNAc-specific receptor when compared to VR.L. These two vesicle populations appear to arise from intracellular organelles which participate in receptor-ligand segregation in rat hepatocytes. Pulse-chase experiments indicate that ligand passes from VR.L to VL. VR.L and VL are also detected in hepatocytes incubated in buffers containing physiologic amounts of Na+; however, the proportion of ligand found in VL is less than in cells incubated in K+-containing buffer. The primary effect of high K+ buffer is to prevent exit of ligand from VL whereas the accumulation of ligand in VR.L is likely secondary to the effect on VL. Membrane protein constituents of VR.L and VL were identified by vectorial lactoperoxidase labeling using a galactosyl conjugate of lactoperoxidase. Vesicles containing Gal-lactoperoxidase were isolated and labeling initiated by addition of 125I, glucose, and glucose oxidase. The labeling patterns for VR.L and VL by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were distinct from the more complex labeling pattern obtained at the cell surface. Analysis by two-dimensional electrophoresis demonstrated a highly selective labeling pattern with only a small number of differences between VR.L and VL. This suggests that the major membrane components of the compartments prior to and following receptor-ligand segregation are the same. Thus, receptors may be selectively removed from these membranes during the process of receptor-ligand segregation.