The traffic patterns established by transport vesicles and other membrane carriers are of fundamental importance for protein localization, modification, and function within eukaryotic cells. The initial contact between transport vesicles and their membrane targets appears to require one of a set of eight or more large hetero-oligomeric `tethering'complexes. This grant proposal focuses on two such complexes, conserved from yeast to mammals, called the conserved oligomeric Golgi (COG) complex and the Dsl1p complex. Both mediate the tethering of COPI vesicles in the early secretory pathway. COG functions in the transport of COPI vesicles within the Golgi apparatus and is therefore essential for normal Golgi complex structure and function. COG defects give rise to congenital disorders of glycosylation. The Dsl1p complex is important for COPI vesicle transport from the Golgi to the ER, a pathway essential for the recycling of the anterograde transport machinery and the retrieval of ER resident proteins. A deeper mechanistic understanding of multisubunit tethering complexes depends critically on determining their three-dimensional structures. During the initial funding period for this grant, we mapped the overall architecture of the COG and Dsl1p complexes and determined the structures or partial structures of four of their subunits. We now propose to tackle larger subassemblies and, in the case of Dsl1p, the entire complex. A second major goal of this proposal is to initiate structural characterization of the interactions between the COG and Dsl1p complexes and other components of the trafficking machinery. The models resulting from such efforts will provide a foundation for generating more incisive mechanistic hypotheses regarding these, and perhaps other, multisubunit tethering complexes. To accomplish these goals, we propose the following specific aims. In the first aim, we will undertake structural analysis of major COG subassemblies, guided by our previous analysis of COG subunit connectivity. Structures of these subassemblies will be elucidated using a combination of x-ray crystallography and EM. We will also conduct an unbiased search for COG-interacting proteins using a highly optimized mass spectrometry approach. Interactions between COG subunits or subassemblies and functionally validated partners will be investigated using biochemical and structural methods. In the second specific aim, we turn our attention to the Dsl1p complex. We have determined crystal structures representing about 50% (by mass) of the Dsl1p complex. We propose to complete this analysis using x-ray crystallography and EM. Finally, in the third specific aim, we will carry out structure/function studies of Dsl1p complex interactions with SNAREs and coat proteins.

Public Health Relevance

The Golgi apparatus plays a key role in protein sorting and glycosylation within the eukaryotic secretory pathway. Defects in vesicular trafficking to, from, and within the Golgi affect both its structure and function. As a consequence, such defects can have pleiotropic effects on the glycosylation and stability of cell surface proteins, leading to human disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cell Structure and Function (CSF)
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Shapiro, Bert I
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Princeton University
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Baker, Richard W; Hughson, Frederick M (2016) Chaperoning SNARE assembly and disassembly. Nat Rev Mol Cell Biol 17:465-79
Ha, Jun Yong; Chou, Hui-Ting; Ungar, Daniel et al. (2016) Molecular architecture of the complete COG tethering complex. Nat Struct Mol Biol 23:758-60
Suckling, Richard J; Poon, Pak Phi; Travis, Sophie M et al. (2015) Structural basis for the binding of tryptophan-based motifs by ?-COP. Proc Natl Acad Sci U S A 112:14242-7
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
Rogers, Jason V; McMahon, Conor; Baryshnikova, Anastasia et al. (2014) ER-associated retrograde SNAREs and the Dsl1 complex mediate an alternative, Sey1p-independent homotypic ER fusion pathway. Mol Biol Cell 25:3401-12
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
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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

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