Endocytosis of mannose‐6‐phosphate binding sites by mouse T‐lymphoma cells

The endocytosis and intracellular transport of mannose‐6‐phosphate conjugated to bovine serum albumin (Man‐6‐P:BSA) by mouse T‐lymphoma cells were investigated in detail using several methods of analysis, both morphological and biochemical. Man‐6‐P:BSA was labeled with fluorescein or 125 I and used to locate both surface and intracellular Man‐6‐P binding sites by light or electron microscopy, respectively. Incubation of cells with either fluorescent or 125 I‐labeled Man‐6‐P:BSA at 0°C revealed a uniform distribution of the Man‐6‐P binding sites over the cell surface. Competition experiments indicate that the Man‐6‐P:BSA binding sites on the cell surface are the same receptors that can recognize lysosomal hydrolases. After as little as 1 min incubation at 37°C, endocytosis of Man‐6‐P binding sites was clearly observed to occur through regions of the plasma membrane and via vesicles that also bound anticlathrin antibody. After a 5–15‐min incubation of cells at 37°C, the internalized ligand was detected first in the cis region of the Golgi apparatus and then in the Golgi stacks using both autoradiography and immunocytochemistry to visualize the ligand. The appearance of Man‐6‐P:BSA in the Golgi region after 15–30 min was confirmed by subcellular fractionation, which demonstrated an accumulation of Man‐6‐P:BSA in light membrane fractions that corresponded with the Golgi fractions. After a 30‐min incubation at 37°C, the internalized Man‐6‐P binding sites were localized primarily in lysosomal structures whose membrane but not lumen co‐stained for acid phosphatase. These results demonstrate a temporal participation of clathrin‐containing coated vesicles during the initial endocytosis of Man‐6‐P binding sites and that one step in the Man‐6‐P:BSA transport pathway between plasma membrane and the lysosomal structure can involve a transit through the Golgi stacks.