Date of Award

Spring 1-1-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Greg Odorizzi

Second Advisor

Corrie Detweiler

Third Advisor

Gia Voeltz

Fourth Advisor

Jingshi Shen

Fifth Advisor

Santiago Di pietro

Abstract

Endosomal sorting complexes required for transport (ESCRTs) function at late endosomal multivesicular bodies (MVBs) to sort ubiquitinated transmembrane proteins into intraluminal vesicles (ILVs) prior to fusion with lysosomes, or the homologous organelle in yeast, the vacuole. There are four distinct ESCRT complexes (ESCRT-0, -I, -II, and -III) that each transiently associate with the cytosolic surface of endosome membranes without getting consumed by the forming ILV. ESCRT-0, -I, and -II each contain one or more subunits that bind ubiquitin to collectively sequester cargo into microdomains on the membrane. Were as ESCRT-0, -I, and -II are constitutively assembled complexes, electrostatic interactions within individual ESCRT-III subunits maintain the proteins as monomers in the cytosol. Autoinhibition of the ESCRT-III subunit Vps20 is relieved after binding ESCRT-II at endosomes, when then nucleates homo-polymerization of the most abundant protein of the ESCRT-III complex, Snf7. Cargo deubiquitination must precede membrane scission to replenish free ubiquitin levels available to the cell, and Doa4 is the hydrolase in S. cerevisiae to do this. I first show that ESCRT-III polymerization can be uncoupled from ILV formation if the cargo-sorting arm of the ESCRT pathway is disabled. I later show that Doa4 has an additional, non- catalytic function that stalls Snf7 depolymerization, and hence, ILV scission. Activated Vps20 directly binds Doa4 to inhibit both its catalytic and membrane scission stalling functions. My data provides insight into the relationship between cargo sorting, cargo deubiquitination, ESCRT-III assembly/ disassembly cycles, and ILV scission.

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Biology Commons

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