? DESCRIPTION (provided by applicant): Endosomal Sorting Complexes Required for Transport (ESCRTs) is critical for a number of fundamental cellular processes including: degradation of transmembrane proteins through the formation of multivesicular bodies (MVBs) during endocytic trafficking, abscission during cytokinesis, budding of enveloped viruses, and biogenesis of some exosomes. Perturbations of ESCRT function have been linked with promoting tumorigenesis and neurological diseases, including hereditary spastic paraplegia and frontotemporal dementia. Dissecting ESCRT function is therefore critical for understanding cellular physiology in both normal and disease states. ESCRTs were originally identified in Saccharomyces cerevisiae, and yeast MVB sorting serves as the best understood model with which to dissect ESCRT function. MVB sorting can be broken into two steps: the recognition/sequestration of cargoes into microdomains, and the budding of these microdomains into the lumen of the endosome to form intralumenal vesicles (ILVs). The canonical signal for cargo inclusion into an ILV is covalent modification with ubiquitin (Ub). Ub binding domains within the early ESCRTs (subcomplexes termed ESCRT-0, -I, and -II) concentrate Ub-modified cargoes into endosomal microdomains. Early ESCRTs also recruit the late ESCRTs (ESCRT-III and associated factors such as Bro1 and Vps4) responsible for membrane deformation and ILV scission. The AAA-ATPase Vps4 also disassembles ESCRT-III to recycle subunits for additional rounds of function. Early and late ESCRT functions are coordinated to facilitate efficient MVB sorting. One aspect of this coordination is removal of Ub from cargo prior to ILV budding. Bro1 is critical in this process through binding to Ub, recruiting the ubiquitin isopeptidase (DUb) Doa4, and modulating ESCRT-III function via Vps4. Our data indicate that Bro1 can both inhibit and stimulate Vps4 activity and Doa4 can relieve Bro1 inhibitory activity. These observations suggest Bro1 acts as a switch to coordinate early and late ESCRT functions to facilitate flux through the MVB pathway. Our central hypothesis is that Bro1 changes from a negative regulator to a positive regulator of Vps4 during the progression of the sorting reaction to coordinate early and late ESCRT function. This hypothesis will be tested through testing three sub-hypothesis: i) Bro1 inhibits ESCRT-III disassembly to promote cargo transfer to ESCRT-III and into the ILV; ii) Bro1 stimulates Vps4 to accelerate ESCRT- III remodeling and complete ILV budding; and iii) a checkpoint is responsible for converting Bro1 from an inhibitor to an activator of Vps4. Addressing these mechanisms of Bro1 function will provide novel insights into analogous roles for human Bro1 Family members Alix and HD-PTP in extracellular vesicle release, cytokinesis and MVB sorting in human systems.
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