PROJECT SUMMARY/ABSTRACT Intracellular membrane trafficking mediates the intracellular delivery of proteins and lipids. The process is bidirectional and consists of the anterograde (secretory) and retrograde (endocytic) branches. Intracellular membrane trafficking is evolutionary conserved, and its machinery is modular, with functionally homologous components operating on different trafficking steps. Therefore, a detailed understanding of one trafficking step will help in understanding the entire intracellular membrane trafficking process. The Conserved Oligomeric Golgi (COG) complex operates as a vesicular tether for intra-Golgi trafficking. The Golgi is the central hub for protein posttranslational modifications, mostly glycosylation. Consequently, the primary job of the COG is to tether vesicles that recycle resident enzymes and cargo receptors. To achieve its function, the COG interacts with SNAREs, Rabs, and other tethers, but the detailed understanding of these interactions is an enigma that we propose to solve by pairwise probing of COG/partner interactions, their kinetics, and by defining their molecular environment through proximity-labeling studies. Depletion of COG causes accumulation of specific transport intermediates – COG complex dependent (CCD) vesicles that are likely to represent a major class of Golgi vesicles that recycle Golgi enzymes and cargo receptors. Mutations in COG subunits result in congenital disorders of glycosylation (CDG) type II category, which belong to a group of autosomal recessive multi-systemic disorders with several distinguishable symptoms that include global developmental defects and microcephaly. These deficits are often accompanied by neurological and liver impairment. COG-CDG defects are studied in patients’ fibroblasts, which do not represent the most affected tissues; a more flexible cell base model will benefit our progress in developing a cure for this disorder. We hypothesize that a revealing of the molecular basis of COG/partner interactions will help in deciphering the mechanisms of assembly/disassembly of vesicle docking platforms and that a detailed analysis of different populations of CCD vesicles will uncover their specific origin, budding and tethering machinery and a complete set of proteins that traffic in a COG-dependent manner. The development of cell-based models for COG-CDGs will allow us to test the effects of different COG mutations without the need for patient involvement and pave the way for the development of treatment protocols. To test this hypothesis, first, we will characterize in molecular interactions between COG and its key partner proteins (Aim1). Next, we will use a degrone-assisted COG depletion to accumulate, purify and characterize CCD vesicles (Aim2). Finally, we will develop and characterize a novel iPSC-based cellular model for COG CDGs (Aim 3). Success in accomplishing these aims will provide a mechanistic understanding of COG complex function, characterize COG-de...