Biology and Biomedical Sciences: Molecular Cell Biology
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Endosomal sorting complex required for transports: ESCRT) machinery responsible for multivesicular body: MVB) biogenesis is essential for receptor downregulation, viral budding and cytokinesis. ESCRT-III is a large polymer built from related ESCRT-III proteins that is thought to help generate intralumenal vesicles: ILVs) within the MVB. How ESCRT-III functions is poorly understood. Although ESCRT-III assembles on the endosomal membrane, its components are predominantly soluble in the cytoplasm. I found that the transition between these two states is controlled by autoinhibitory domains within ESCRT-III proteins, which I identified by structure/ function analysis in four human ESCRT-III proteins - Charged multivesicular body protein2A: CHMP2A), CHMP3, CHMP6, and CHMP4A. Biochemical and functional assays confirmed that the C-terminally located autoinhibitory domains control cycling between a "closed" state which they are soluble monomers and an "open" state in which they assemble into membrane associated complexes. While searching for cellular factor(s) that might regulate transition between these states, I found that LIP5, a proposed cofactor of the ATPase VPS4, binds efficiently to the autoinhibitory domains of a subset of ESCRT-III proteins including CHMP1B, 2A and 3. Because VPS4 disassembles ESCRT-III complexes, this direct interaction between its cofactor LIP5 and ESCRT-III proteins can enhance VPS4 mediated ESCRT-III disassembly. To ask when and how individual ESCRT-III proteins and VPS4 contribute to ILV formation in cultured cells, I establish reagents to detect and manipulate these proteins including antibodies and effective small interference RNAs. I used these tools to show that representative of two classes of cell surface receptors, epidermal growth factor receptor: EGFR), a tyrosine kinase receptor and delta-opioid receptor: DOR), a G-protein coupled receptor use ESCRT-III and VPS4 to undergo downregulation via lysosomal degradation. Taken together the studies in this thesis provide insights into roles and regulation of ESCRT-III in MVB biogenesis.
Shim, Soomin, "Role And Regulation Of ESCRT-III In Multivesiculr Body Biogenesis" (2009). All Theses and Dissertations (ETDs). 323.