Isolation and preliminary characterization of the major membrane boundaries of the endocytic pathway in lymphocytes

Abstract

Plasma membrane, coated pits, endosomes, and lysosomes were isolated from a mouse T lymphoma cell line using a density shift protocol in which these compartments were selectively loaded with gold conjugates. The plasma membrane was prepared after selective labeling for 1 h at 2 degrees C with gold-ricin and gave a yield of 40% according to enzymatic and antigenic markers. Endosomes were obtained by loading the cells for 2 h at 22 degrees C with gold complexed to an antimouse transferrin receptor mAb. Coated pits were isolated using a similar procedure, but after an incubation at 10 degrees C, which allowed deep invagination of the pits but prevented internalization. The yield (calculated using the recovery of [125I]transferrin) was 32% for endosomes and 10% for coated pits. Finally lysosomes were prepared by loading the cells for 18 h at 37 degrees C with gold low density lipoproteins (LDLs) followed by a 3-h chase at 37 degrees C with LDL alone. The final lysosome yield (based on the recovery of lysosomal enzymes) was 16%. Studies of the protein composition of these cellular compartments on two-dimensional gels showed that while some major proteins are present throughout the pathway, specific proteins can be identified in each of the isolated fractions. The greatest change in the pattern of protein constituents seen along the pathway was between endosomal and lysosomal preparations.