Coated vesicles were purified from human placenta by sedimentation, isopycnic centrifugation, and gel filtration. Quantitative Western blotting of the endogenous transferrin receptor (tfR) demonstrated the presence, on average, of roughly one receptor per vesicle. TfR appeared undersaturated with transferrin. After solubilizing vesicles in nonionic detergent, we looked for evidence of tfR interactions with other proteins. Solubilized tfR had an unexpectedly high mobility by gel filtration, apparently resulting from its self-association. This property was not seen in purified tfR or in tfR from a different cell fraction. The tfR complexes, though noncovalent, were largely resistant to conditions that disassemble coat proteins, and they did not appear to contain any other protein species.

We used a combination of electron microscopy and proteolytic dissection to study the substructure of the clathrin trimer. The fragments of a heavy chain generated by limited proteolysis of cages were examined by rotary shadowing after disassembly. Correlation of lengths and molecular weights allowed us to map certain cleavage points along an arm and to assign them to positions in a model for a cage. We found that a particularly stable fragment of 52,000-59,000 Mr (depending on the enzyme) corresponded to the knob-like terminal domain at the tip of each arm.