Vesicle transport within the Golgi apparatus is a useful system for studying the targeting specificity of intracellular transport vesicles. The mammalian Golgi consists of four to eight cisternae, each of these containing a unique set of protein- and lipid-modifying enzymes. Most of the transport through the Golgi occurs by cisternal maturation, which suggests that secretory proteins always remain within the cisternae, while resident enzymes are recycled via retrograde vesicles. This sorting model implies that several distinct types of retrograde vesicles (with differing enzyme content) generate the Golgi?s differential enzyme distribution. The central coordinator for retrograde vesicle tethering at the Golgi is the evolutionarily conserved oligomeric Golgi (COG) complex. The COG complex consists of eight gene products, each of which is critical for the Golgi functions. The COG complex functions in the tethering of vesicles that recycle Golgi glycosylation enzymes residing in cis, medial, and trans compartments. Consequently, defects in seven COG subunits have been identified in patients with congenital disorders of glycosylation type II. The COG complex is required for a broad range of essential processes, including protein and lipid glycosylation, sorting and retrograde vesicular trafficking, but the exact mechanism of COG function is an enigma. Detailed analyses of HEK293T and HeLa knock-out (KO) cell lines depleted of individual COG subunits have revealed that each subunit is indispensable to the stability and function of the entire COG complex. Further, a complete COG complex is essential for the stability of a subset of Golgi enzymes, but nonessential for another subset, indicating the existence of both COG-dependent and COG-independent Golgi recycling pathways. We propose that the COG complex orchestrates local recycling of a subset of Golgi enzymes via multipronged interaction with specific SNAREs, Rabs, and coiled-coil tethering factors. COG malfunction results in a rapid loss and degradation of COG- dependent vesicles, triggering upregulation of selective compensatory mechanisms (modulation of cholesterol biosynthesis, Golgi perinuclear repositioning, and modification of the endocytic pathway) which are essential for survival of COG-deficient human cells. To test this hypothesis, first we will utilize gene-edited cell lines and a combination of biochemical and microscopy tools to investigate the molecular details of the COG complex? dependent enzyme-recycling pathway and the COG complex?independent pathway (Aim 1). Next, we will reconstitute COG complex vesicle-tethering activity in vitro (Aim 2). Finally, we will investigate novel stress and compensatory mechanisms that allow for the survival of COG-deprived human cells (Aim 3). The existence of several independent Golgi recycling mechanisms is likely to be important for the elasticity of eukaryotic secretory and endocytic pathways. Understanding how the COG complex spatially and temporally controls the precise tethering of selected transport vesicles is critical to our understanding of membrane trafficking and protein glycosylation in human cells.

Public Health Relevance

In all eukaryotic cells, intracellular membrane trafficking is critical for a range of essential cellular functions, including protein sorting and secretion, post-translational modifications, cell signaling, cell polarization, and cell maintenance. Defects in membrane trafficking can underlie or exacerbate a number of human diseases such as cancer, diabetes mellitus, Alzheimer?s disease, cystic fibrosis, Hermansky-Pudlak syndrome, and congenital disorders of glycosylation. We will determine how the key components of the Golgi membrane trafficking machinery work together to direct efficient protein secretion and protein glycosylation in human cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM083144-12
Application #
9920712
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Ainsztein, Alexandra M
Project Start
2008-08-01
Project End
2022-04-30
Budget Start
2020-05-01
Budget End
2021-04-30
Support Year
12
Fiscal Year
2020
Total Cost
Indirect Cost
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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Climer, Leslie K; Hendrix, Rachel D; Lupashin, Vladimir V (2018) Conserved Oligomeric Golgi and Neuronal Vesicular Trafficking. Handb Exp Pharmacol 245:227-247
Blackburn, Jessica B; Kudlyk, Tetyana; Pokrovskaya, Irina et al. (2018) More than just sugars: Conserved oligomeric Golgi complex deficiency causes glycosylation-independent cellular defects. Traffic 19:463-480
Ishii, Midori; Lupashin, Vladimir V; Nakano, Akihiko (2018) Detailed Analysis of the Interaction of Yeast COG Complex. Cell Struct Funct 43:119-127
Morelle, Willy; Potelle, Sven; Witters, Peter et al. (2017) Galactose Supplementation in Patients With TMEM165-CDG Rescues the Glycosylation Defects. J Clin Endocrinol Metab 102:1375-1386
Comstra, Heather S; McArthy, Jacob; Rudin-Rush, Samantha et al. (2017) The interactome of the copper transporter ATP7A belongs to a network of neurodevelopmental and neurodegeneration factors. Elife 6:
Miller, Cheryl N; Smith, Erin P; Cundiff, Jennifer A et al. (2017) A Brucella Type IV Effector Targets the COG Tethering Complex to Remodel Host Secretory Traffic and Promote Intracellular Replication. Cell Host Microbe 22:317-329.e7
Hadden, Coedy; Fahmi, Tariq; Cooper, Anthonya et al. (2017) Serotonin transporter protects the placental cells against apoptosis in caspase 3-independent pathway. J Cell Physiol 232:3520-3529
Potelle, Sven; Dulary, Eudoxie; Climer, Leslie et al. (2017) Manganese-induced turnover of TMEM165. Biochem J 474:1481-1493
Siegel, Nisan; Lupashin, Vladimir; Storrie, Brian et al. (2016) High-magnification super-resolution FINCH microscopy using birefringent crystal lens interferometers. Nat Photonics 10:802-808

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