Project Summary/Abstract One third of eukaryotic proteins transit the secretory pathway for sorting to specific locations, including the endoplasmic reticulum (ER), Golgi, plasma membrane or extracellular milieu. Since misdirected proteins cannot function, the secretory pathway is critical for establishing and maintaining normal cell and tissue physiology. The COPII coat protein complex, which mediates anterograde trafficking from the ER, is a key control point for protein targeting. Indeed, mutations in COPII genes cause a range of human diseases, including cranio- lenticulo-sutural dysplasia (CLSD), a subtype of osteogenesis imperfecta (OI), hematologic disorders and myriad neurological defects. Detailed knowledge of COPII trafficking is required to understand its role in cell physiology and to treat disorders in which it is disrupted. However, while the core COPII machinery is well defined, little is known about how vertebrate cells regulate COPII activity in response to normal or pathological signals or stress. We and others have found that several COPII proteins are modified by O-linked b-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation. At the start of the prior project period, the effects of O-GlcNAcylation on COPII remained almost entirely unknown. Since then, we have defined the scope of O-GlcNAcylation in the core COPII system, identified functional effects of O-GlcNAc cycling in vesicle trafficking, devised new quantitative glycoproteomics methods to profile O-GlcNAc changes in response to secretory pathway stress and other stimuli, and demonstrated that particular Sec23A O-GlcNAc sites are required for endogenous collagen trafficking in cultured human cells and in the chondrocytes of developing zebrafish. Together, these results demonstrate that site-specific O-GlcNAcylation of COPII proteins governs cargo trafficking in vertebrate cells and tissues. However, major unanswered questions remain, including the mechanistic effects that O-GlcNAc exerts on COPII proteins, the upstream stimuli that modulate COPII O- GlcNAcylation and the global landscape of O-GlcNAc signaling in the early secretory pathway. Here, we propose to address these important questions in the next award period. In Aim 1, we will dissect the molecular mechanisms by which O-GlcNAc cycling influences COPII vesicle trafficking. In Aim 2, we will identify upstream stimuli that control COPII protein O-GlcNAcylation and determine the downstream effects of this signaling in protein secretion and cell cycle progression. In Aim 3, we will use our glycoproteomics methods to canvass proteome-wide O-GlcNAc signaling in response to COPII cargo trafficking, and provide an integrated picture of crosstalk between O-GlcNAcylation and phosphorylation in protein secretion. Our work will shed new light on how O-GlcNAc regulates trafficking in cells and tissues, and may reveal new opportunities to treat diseases of COPII dysfunction by manipulating ...