Hirst, Jennifer; Isolation and characterisation of the multivesicular body and its role in the trafficking of membrane proteins. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Multivesicular bodies (MVB) were first described in rat ovum as spherical vacuoles 200-800nm in diameter, containing small internal vesicles of 40–80nm. Detailed morphological studies have since identified MVBs in numerous cell lines. Although the role of MVBs as an intermediate compartment of the endocytic pathway is clear, as demonstrated by labeling with endocytosed molecules, its function in endocytosis is unclear. This is in part hampered by discrepancies in endosome nomenclature, and the inability to isolate endosomes to high purity. Using an internalised antibody to epidermal growth factor receptor or transferring receptor complexed to colloidal gold, to modify endosome density, I have been able to isolate a highly purified MVB preparation from HEp.2 cells. The ability to purify MVBs away from plasma membrane and other smooth membranes permitted the detailed biochemical and morphological investigation of the physical and functional characteristics of the MVB: A monoclonal antibody against a 70kD protein was raised against isolated MVB fractions and the antigen shown to partially co-localise with an internalised marker of the MVB. Further, using pulse chase procedures I demonstrate that the MVB is the primary delivery site of newly synthesised cation-independent mannose 6-phosphate receptor and the lysosomal hydrolase, cathepsin D, from the trans-Golgi Network. The subsequent rapid secretion of cathepsin D following arrival in the MVB also implies a direct trafficking route from the MVB to the plasma membrane.

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