Subfractionation of the endocytic pathway: isolation of compartments involved in the processing of internalised epidermal growth factor-receptor complexes

• Published in: Journal of cell science Vol. 94; no. 4; pp. 685 - 694
• Main Authors: FUTTER, C. E, HOPKINS, C. R
• Format: Journal Article
• Language: English
• Published: Cambridge Company of Biologists 01.12.1989
• Subjects: Biological and medical sciences; Cell physiology; Endocytosis; Endocytosis - physiology; Epidermal Growth Factor - physiology; ErbB Receptors - physiology; Fundamental and applied biological sciences. Psychology; Humans; Microscopy, Electron; Molecular and cellular biology; Receptors, Transferrin - physiology; transferrin; Transferrin - physiology
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.94.4.685

The aim of the present study was to isolate different parts of the endocytic pathway in order to examine the role of epidermal growth factor (EGF)-receptor internalisation in mediating the biological effects of EGF. We have used an antibody to the transferrin receptor complexed with colloidal gold to modify the density of the endocytic compartments so that they can be purified by sucrose density centrifugation. Using this technique, we have been able to isolate a highly purified preparation of endocytic vesicles from H.Ep.2 cells that contain internalised EGF. By employing pulse--chase protocols, it is possible to isolate the different parts of the endocytic pathway and show that they are temporally distinct with regard to the processing of EGF. It should now be possible to examine interactions between the EGF receptor and intracellular substrates in different parts of the endocytic pathway.