The traffic patterns established by transport vesicles and other membrane carriers are of fundamental importance for protein localization, modification, and function within eukaryotic cells. Vesicle docking and fusion requires, in addition to soluble NSF attachment protein receptors (SNAREs), multisubunit tethering complexes (MTCs). This proposal focuses on three MTCs, conserved from yeast to mammals: the Dsl1 complex, the conserved oligomeric Golgi (COG) complex, and the homotypic fusion and vacuole protein sorting (HOPS) complex. The Dsl1 complex functions in COPI vesicle transport from the Golgi apparatus to the endoplasmic reticulum (ER), a pathway essential for the recycling of the anterograde transport machinery and the retrieval of ER-resident proteins. The COG complex functions in retrograde transport within the Golgi. As a result, COG is essential for normal Golgi structure and function, and defects in COG give rise to congenital disorders of glycosylation. Finally, membrane fusion at late endosomes and lysosomes/vacuoles depends on the HOPS complex. All six subunits of human HOPS are among the seven host proteins recently discovered to be required for Marburg and Ebola virus entry. We hypothesize that MTCs, through interactions with Rabs, SNAREs, Sec1/Munc18 proteins, vesicle coat proteins, and phospholipids, function to orchestrate the docking and fusion of transport vesicles. Achieving a deeper mechanistic understanding of MTC function depends critically on elucidating their structures and determining how they interact with other elements of the trafficking machinery. To this end, we propose three specific aims.
In Aim 1, we will characterize functional interactions between the Dsl1 complex and other trafficking factors using x-ray crystallography and single particle electron microscopy (EM). In addition, we will capitalize on our complete structure of the Dsl1 complex by designing mutants to use in proteomic and synthetic genetic screens for additional Dsl1-interacting partners.
In Aim 2, we will use single-particle EM to complete our mapping of the eight different subunits into the overall structure of the COG complex, complemented by x-ray crystallographic studies of interacting elements within COG sub- assemblies. Furthermore, we will determine COG-SNARE complex structures in order to elucidate how COG guides SNARE assembly. Finally, in Aim 3 we propose an entirely new project, structural studies of the HOPS complex and its interaction with SNAREs. We will determine structures of key HOPS subunits and sub- assemblies, which can then serve as blueprints for in vivo and in vitro functional studies. In addition, as in the first two Aims, w will use x-ray crystallography to study complexes with SNAREs. Because the HOPS complex is unrelated to the Dsl1 and COG complexes, this work should reveal both class-specific differences and common principles among MTCs, thereby deepening our mechanistic understanding of these fascinating components of the intracellular trafficking machinery.

Public Health Relevance

The proposed work entails structural studies of the machinery responsible for the sorting and transport of proteins and lipids among the compartments of a eukaryotic cell. Mutations in many components of this machinery, by upsetting cellular architecture and function, lead to human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM071574-09
Application #
8512293
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2005-03-01
Project End
2017-02-28
Budget Start
2013-06-01
Budget End
2014-02-28
Support Year
9
Fiscal Year
2013
Total Cost
$337,167
Indirect Cost
$121,696
Name
Princeton University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
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Baker, Richard W; Jeffrey, Philip D; Zick, Michael et al. (2015) A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly. Science 349:1111-4
Ha, Jun Yong; Pokrovskaya, Irina D; Climer, Leslie K et al. (2014) Cog5-Cog7 crystal structure reveals interactions essential for the function of a multisubunit tethering complex. Proc Natl Acad Sci U S A 111:15762-7
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Baker, Richard W; Jeffrey, Philip D; Hughson, Frederick M (2013) Crystal Structures of the Sec1/Munc18 (SM) Protein Vps33, Alone and Bound to the Homotypic Fusion and Vacuolar Protein Sorting (HOPS) Subunit Vps16*. PLoS One 8:e67409
Bharucha, Nike; Liu, Yang; Papanikou, Effrosyni et al. (2013) Sec16 influences transitional ER sites by regulating rather than organizing COPII. Mol Biol Cell 24:3406-19
McMahon, Conor; Studer, Sean M; Clendinen, Chaevia et al. (2012) The structure of Sec12 implicates potassium ion coordination in Sar1 activation. J Biol Chem 287:43599-606
Ren, Qiansheng; Wimmer, Christian; Chicka, Michael C et al. (2010) Munc13-4 is a limiting factor in the pathway required for platelet granule release and hemostasis. Blood 116:869-77
Yen, Wei-Lien; Shintani, Takahiro; Nair, Usha et al. (2010) The conserved oligomeric Golgi complex is involved in double-membrane vesicle formation during autophagy. J Cell Biol 188:101-14

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