Endosomes segregate endocytosed macromolecules destined to be degraded in the lysosome from molecules that are either recycled back to the cell surface or routed to other intracellular organelles. The multivesicular body (MVB) is a late endosome that contains vesicles formed by invagination of the endosomal membrane toward the compartmental lumen. Many growth factor receptors and stimulatory components of the immune system are sorted into MVB vesicles. Furthermore, human immunodeficiency virus-1 and other enveloped RNA viruses usurp components required for MVB vesicle formation in order to produce virions, thereby enabling the spread of viral infection. The objective of this application is to determine the molecular basis of MVB vesicle-mediated transport. Previous work on this project has identified mechanisms by which the Bro1 protein in Saccharomyces cerevisiae participates in the MVB cargo sorting pathway. The proposed work seeks to expand the scope of this analysis to understand the molecular mechanisms that regulate MVB vesicle formation. This process is impaired in cells lacking functional endosomal sorting complexes required for transport (ESCRTs) as a result of endosomes forming `class E compartments.'Preliminary studies suggest that class E compartment formation is driven by the accumulation of ubiquitinated proteins at endosomes and depends on endosomal tethering complexes.
Specific aim 1 will determine the mechanism that regulates protein deubiquitination at endosomes, specific aim 2 will explore how ubiquitinated proteins and tethering components cooperate to create class E compartments, and specific aim 3 will investigate the mechanism that regulates polymerization of Snf7, which has been implicated as the driving force of MVB vesicle budding. All three specific aims will be pursued using a combination of genetic, biochemical, and microscopic studies to examine a variety of mutant yeast strains.

Public Health Relevance

The proposed project seeks to understand the mechanistic regulation of the yeast ESCRT machinery, which is highly related to the ESCRT machinery that functions in human cells. In humans, the ESCRT machinery functions to downregulate many receptor-activated signal transduction pathways that control cell division, and tumorigenesis has been linked to mutations that disrupt ESCRT activity, making components of the ESCRT machinery candidate targets for gene therapy. The ESCRT machinery also is usurped by the human immunodeficiency virus-1 for the production of infectious virions, making components of the ESCRT machinery candidate targets for anti-viral therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM065505-09S1
Application #
8714615
Study Section
Cell Structure and Function (CSF)
Program Officer
Ainsztein, Alexandra M
Project Start
2003-09-01
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
9
Fiscal Year
2013
Total Cost
$97,212
Indirect Cost
$31,872
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
007431505
City
Boulder
State
CO
Country
United States
Zip Code
80309
Chi, Richard J; Liu, Jingxuan; West, Matthew et al. (2014) Fission of SNX-BAR-coated endosomal retrograde transport carriers is promoted by the dynamin-related protein Vps1. J Cell Biol 204:793-806
Babst, Markus; Odorizzi, Greg (2013) The balance of protein expression and degradation: an ESCRTs point of view. Curr Opin Cell Biol 25:489-94
Richter, Caleb M; West, Matthew; Odorizzi, Greg (2013) Doa4 function in ILV budding is restricted through its interaction with the Vps20 subunit of ESCRT-III. J Cell Sci 126:1881-90
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
McMurray, Michael A; Stefan, Christopher J; Wemmer, Megan et al. (2011) Genetic interactions with mutations affecting septin assembly reveal ESCRT functions in budding yeast cytokinesis. Biol Chem 392:699-712
Kallay, Laura M; Brett, Christopher L; Tukaye, Deepali N et al. (2011) Endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44 functions independently and downstream of multivesicular body formation. J Biol Chem 286:44067-77
Nickerson, Daniel P; West, Matthew; Henry, Ryan et al. (2010) Regulators of Vps4 ATPase activity at endosomes differentially influence the size and rate of formation of intralumenal vesicles. Mol Biol Cell 21:1023-32
Haas, Thomas J; Sliwinski, Marek K; Martinez, Dana E et al. (2007) The Arabidopsis AAA ATPase SKD1 is involved in multivesicular endosome function and interacts with its positive regulator LYST-INTERACTING PROTEIN5. Plant Cell 19:1295-312
Richter, Caleb; West, Matthew; Odorizzi, Greg (2007) Dual mechanisms specify Doa4-mediated deubiquitination at multivesicular bodies. EMBO J 26:2454-64
Nickerson, Daniel P; West, Matthew; Odorizzi, Greg (2006) Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes. J Cell Biol 175:715-20

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