Using a cell-free system than mimics the mitotic cycle of the Golgi apparatus, we have been able to identify many of the proteins involved in the fragmentation and reassembly of this organelle. The last granting period focused on the reassembly of Golgi cisternae that is catalyzed by two ATPases, NSF and p97. NSF was shown to have an activity additional to its well-characterized role in unraveling SNARE complexes. It was found to add the ubiquitin-like protein, GATE-16, to the v-SNARE, GOS-28, a process that prevented the formation of non-productive cis SNARE complexes, and primed the SNARE for its interaction with the cognate t-SNARE, syntaxin-5. The p97 ATPase utilizes p47 as an adaptor molecule and this was shown to recognize mono-ubiquitinated proteins as part of the Golgi reassembly process. Other adaptor molecules were identified and characterized, notably the Ufd1p/Np14 complex, and this was shown to mediate p97 action in processes ranging from ER-associated degradation through to nuclear envelope reassembly. The present proposal continues the analysis of these two ATPase-driven pathways focusing on the following aims: 1: Studying the p115 tethering protein to work out precisely how it choreographs the capture and docking of cis-directed COPI transport vesicles. 2: Characterizing a new tethering complex that likely mediates the capture and docking of other COPI vesicles to medial/trans cistemae. The composition and function of these vesicles will also be characterized as will others identified and isolated through capture by different tethers. 3: Determining the role played by ubiquitin in the p97 pathway of Golgi reassembly. Ubiquitinated targets will be identified and characterized. 4: Testing the idea that p97 unravels t-t SNARE complexes just as NSF unravels v-t SNARE complexes. The fusion of ER membranes in budding yeast will be used as the assay. Though the main thrust of this application is the study of fundamental membrane traffic processes, there are medical implications. The tethering proteins were first identified as auto-antigens in patients with Sjogren's syndrome, and in one case as a partner for OCRL1, a PIP2 phosphatase, implicated in oculocerebrorenal syndrome. Ufd1p is mutated in DiGeorge syndrome, a congenital developmental disorder, and the role played by p97 in unraveling protein aggregates has implicated this ATPase in neurodegenerative diseases ranging from Alzheimer's to Huntington disease. Insights into the molecular mechanism of Golgi reassembly may therefore provide insight into these medical conditions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060919-08
Application #
7219534
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Shapiro, Bert I
Project Start
2000-03-01
Project End
2008-09-30
Budget Start
2007-04-01
Budget End
2008-09-30
Support Year
8
Fiscal Year
2007
Total Cost
$312,536
Indirect Cost
Name
Yale University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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