The Biochemistry and physiology of the endocytic apparatus

Cells have evolved a number of specialized mechanisms to transport molecules across the plasma membrane barrier. Endocytic processes involve the invagination and internalization of membrane vesicles and their contents. Conversely, secretory events involve fusion of membrane vesicles with the plasma membrane. The net effect of such activities is the mixing of intracellular and plasma membrane components. Cells must therefore posses some type of sorting mechanism to counteract this randomization and it is believed that the endocytic pathway plays a part in sorting internalized membrane and its constituents. The present study made use of a specific tracer molecule to analyze the compartments involved in the endocytosis/recycling pathway. Transferrin and the transferrin receptor were used to study the endocytic pathway because this ligand along with its receptor traverses the entire endocytic and recycling pathway without being degraded. Transferrin was chemically coupled to horseradish peroxidase in order to specifically target peroxidase activity to the endocytic apparatus. Peroxidase can be used to alter the physical properties of compartments containing the enzyme by affecting an increase in buoyant density, and a crosslinking of luminal contents. The density shift procedure was used to demonstrate that transferrin, low density lipoprotein, and epidermal growth factor are internalized into the same endocytic compartment. A combination of the density shift and crosslinking reactions was used to determine that the endocytic pathway responsible for the accumulation of ligand does not include the Golgi apparatus. Use of the conjugate also provided a means to study the movement of unoccupied receptors. It was determined that intracellular transferrin receptors in HeLa cells are the result of the constitutive internalization of unoccupied receptors. It was further demonstrated that endocytic compartments internalized at different times remain temporally segregated. These results suggest that one way in which surface receptor number can be regulated is the by rate of internalization of unoccupied receptors. The results of the present studies provide a quantitative approach to describing the nature and constituents of the endocytic/recycling pathway. They provide strong support for the notion that the endosome is the intracellular organelle responsible for sorting different receptor/ligand complexes and raise the possibility that this organelle may also participate in the regulation of plasma membrane proteins. Further, these studies provide a method and conceptual approach for further experimentation on the role of the endosome in cellular physiology and metabolism.