Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology

Language

English (en)

Date of Award

January 2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Phyllis Hanson

Abstract

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.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7D50K0Q

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