This work is designed to further our understanding of membrane trafficking in eukaryotic cells. Intracellular membrane trafficking is critically important to cellular and organismal development. Furthermore, various malfunctions in vesicle tethering factors, such as the Conserved Oligomeric Golgi (COG) complex, have a significant impact on processes such as protein sorting, glycosylation and organelle integrity. We propose a detailed structural-functional analysis of the machinery that determines high-fidelity bidirectional protein and lipid trafficking in the Golgi apparatus. The functional interplay between SNAREs, Rabs and tethering factors will be investigated;we also hope to identify the sequence of events that lead to vesicle docking and fusion. Our long-term goal is to establish the molecular mechanism through which intra-Golgi membrane trafficking is accomplished. The COG complex is an eight subunit peripheral Golgi membrane hetero-oligomeric protein complex that is organized into lobes A (Cog2-4) and B (Cog5-7) with Cog1p and Cog8p bridging these lobes. The COG complex is thought to play a critical role in vesicle tethering processes involving retrograde Golgi transport of resident proteins responsible for sugar chain (glycan) biosynthesis. Based on data from our and other laboratories, we proposed a model for COG complex function in which COG complex specifically tethers vesicles that retrieve recycling cis/medial-Golgi proteins from trans-Golgi compartments. We hypothesize that the COG complex orchestrates vesicle tethering and fusion through a cascade of protein-protein interactions. These interactions include transient contacts with coil-coil tethering factors and specific Rab and SNARE molecules. The goal of this proposal is to define both the sequence and the molecular mechanism of human COG complex interactions with core components of intra-Golgi docking and fusion machinery. We will accomplish this goal through (1) investigation the molecular details of interaction between the COG complex and intra-Golgi SNARE proteins, (2) characterization the molecular basis by which Golgi Rab proteins interact with the COG complex, (3) characterization the processes of COG complex self-assembly and membrane attachment and (4) characterization the functional interplay between the COG complex, coil-coil tethering factor(s), Rabs and SNAREs. This proposal is integrated methodologically because we will apply various cell biological, biochemical and microscopic tools to approach the problem. We anticipate that proposed experiments would test our model of COG complex function and explain cellular defects occurring in cells after both acute and permanent depletion of COG complex activity. Public Health Relevance: In humans, mutations in COG complex subunits alter the Golgi organization and intracellular membrane trafficking (Ungar et al., 2002;Zolov and Lupashin, 2005;Shestakova et al., 2006;Ungar et al., 2006), resulting in disruption of multiple glycosylation pathways, and, ultimately in suffering from psychomotor retardation and hypotonia (COG1-mutant (Foulquier et al., 2006), and COG8-mutant (Foulquier et al., 2007) patients) or in lethality from cardiac insufficiency and severe liver disease (COG7-mutant (Wu et al., 2004) patients). We anticipate that proposed experiments would test our model for the COG complex function, provide explanation for cellular defects occurring in patients with altered COG complex activity and provide molecular basis for development treatments for the COG complex-related deceases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083144-05
Application #
8272541
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2008-08-01
Project End
2013-11-30
Budget Start
2012-06-01
Budget End
2013-11-30
Support Year
5
Fiscal Year
2012
Total Cost
$357,980
Indirect Cost
$111,164
Name
University of Arkansas for Medical Sciences
Department
Physiology
Type
Schools of Medicine
DUNS #
122452563
City
Little Rock
State
AR
Country
United States
Zip Code
72205
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