Multivesicular body (MVB) sorting is a critical process within the endocytic pathway wherein portions of the endosomal membrane bud into the endosomal lumen. Failure to target activated growth factor receptors into the MVB pathway results in prolonged signaling that can contribute to tumorigenesis and defects in organism development. Defects in MVB targeting of the epithelial sodium channel result in an inherited form of hypertension (Liddle's syndrome). Neurodegenerative diseases have been linked to dysfunction of the MVB pathway, and aberrant trafficking of lipids through this pathway also contributes to a number of human disease states, including atherosclerosis. MVB sorting is mediated by the endosomal sorting complexes required for transport (ESCRT-0, I, II, and III) and associated factors including the AAA-ATPase Vps4. In addition to roles in MVB sorting, the ESCRTs and Vps4 are usurped by enveloped viruses (e.g. HIV-1 and Ebola) to execute their cellular egress. Furthermore, the ESCRTs and Vps4 contribute to membrane abscission during cytokinesis. These examples highlight numerous processes impacted by ESCRT function as well as maladies that arise upon dysfunction. Vps4 and ESCRT-III act in a coordinated manner during membrane deformations of similar topology (away from the cytoplasm) that occur in MVB sorting, viral budding, and membrane abscission. Dissociation of ESCRT-III through Vps4 ATP hydrolysis is required for these processes, while ESCRT-III also regulates Vps4. The studies presented in this proposal apply biochemical approaches to probe the enzymatic activity of Vps4, the regulation of Vps4 by ESCRT-III, and the mechanism by which Vps4-stimulated ESCRT-III dissociation facilitates intralumenal vesicle formation. These inquiries are complemented by in vivo functional analyses in the model organism Saccharomyces cerevisiae. Completion of this experimental plan will provide insight into the conserved evolutionary function of ESCRT-III and Vps4 in membrane constriction during MVB sorting, viral budding and cytokinesis.

Public Health Relevance

Multivesicular body sorting machinery deforms the membrane in a unique manner to permit growth factor receptor downregulation, cellular division and viral budding. Understanding how this process takes place will lead to insights into the prevention of cancer, neurodegenerative diseases and hypertension as well as mechanisms of viral reproduction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM073024-09
Application #
8473224
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Ainsztein, Alexandra M
Project Start
2005-02-01
Project End
2014-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
9
Fiscal Year
2013
Total Cost
$317,890
Indirect Cost
$116,311
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
Davies, Brian A; Norgan, Andrew P; Payne, Johanna A et al. (2014) Vps4 stimulatory element of the cofactor Vta1 contacts the ATPase Vps4 ?7 and ?9 to stimulate ATP hydrolysis. J Biol Chem 289:28707-18
Norgan, Andrew P; Davies, Brian A; Azmi, Ishara F et al. (2013) Relief of autoinhibition enhances Vta1 activation of Vps4 via the Vps4 stimulatory element. J Biol Chem 288:26147-56
Shestakova, Anna; Curtiss, Matt; Davies, Brian A et al. (2013) The linker region plays a regulatory role in assembly and activity of the Vps4 AAA ATPase. J Biol Chem 288:26810-9
Wemmer, Megan; Azmi, Ishara; West, Matthew et al. (2011) Bro1 binding to Snf7 regulates ESCRT-III membrane scission activity in yeast. J Cell Biol 192:295-306
Babst, Markus; Davies, Brian A; Katzmann, David J (2011) Regulation of Vps4 during MVB sorting and cytokinesis. Traffic 12:1298-305
Shestakova, Anna; Hanono, Abraham; Drosner, Stacey et al. (2010) Assembly of the AAA ATPase Vps4 on ESCRT-III. Mol Biol Cell 21:1059-71
Davies, Brian A; Azmi, Ishara F; Payne, Johanna et al. (2010) Coordination of substrate binding and ATP hydrolysis in Vps4-mediated ESCRT-III disassembly. Mol Biol Cell 21:3396-408
Lee, Jacqueline R E; Oestreich, Andrea J; Payne, Johanna A et al. (2009) The HECT domain of the ubiquitin ligase Rsp5 contributes to substrate recognition. J Biol Chem 284:32126-37
Xiao, Junyu; Chen, Xiao-Wei; Davies, Brian A et al. (2009) Structural basis of Ist1 function and Ist1-Did2 interaction in the multivesicular body pathway and cytokinesis. Mol Biol Cell 20:3514-24
Davies, Brian A; Lee, Jacqueline R E; Oestreich, Andrea J et al. (2009) Membrane protein targeting to the MVB/lysosome. Chem Rev 109:1575-86

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