PROJECT SUMMARY AIM1 studies how a therapeutic sugar is mobilized for glycosylation. Sugars taken up from outside the cell are handled differently than those produced inside the cell. We found that activated fucose (GDP-Fuc) exists in multiple cytoplasmic pools that differentially contribute to glycosylation., There is no homogenous, feely mixing pool of sugars. This segregation requires a previously unimagined level of cellular organization to distinguish fucose from different origins. We measured simultaneous contributions of the de novo, exogenous, and recycled (salvaged) fucose into multiple glycoproteins. Their contributions varied based on the fucose origin, glycoprotein acceptor, cellular compartment (ER or Golgi), and fucosyltransferase. These multiple, separate, yet conversant, GDP-fucose pools require regulation, since their combined amount remains constant. We will use multiple 13C- sugar labeling and quantitative lectin binding as functional readouts to probe this cellular organization and control. Now we will determine 1.) how the metabolic origin of fucose affects its ultimate incorporation into different glycoproteins. 2.) basis of fucose segregation 3). the effect of relocating fucosyltransferses from the ER to the Golgi. 4.) the genes and transporters involved in fucose segregation, degradation, and recycling. 5.) Use lectin-based siRNA knockdown and whole genome CRISPR screens to search for genes that impact GDP-Fuc pool utilization. Discovery of multiple GDP-Fucose pools led us to find evidence for separate GDP-Mannose pools. We will measure functional effects using 13C-monosaccharide incorporation into bulk N- and O-linked glycans or into single glycoproteins using lectin binding. Finding the players and mechanisms that segregate pools will likely apply to other monosaccharides and precursors for other macromolecules. In AIM 2, we will continue to identify and validate new CDGs linking clinical medicine to cell biology and glycosylation. We already identified 25 types of CDG. Each candidate requires individualized biochemical and functional validation. Our decades-long clinical pipeline provides a steady stream of potential candidate genes. We prioritize them based on inheritance pedigrees, nature of the variants, interactome and structural analysis of the protein. UGGT1, GOLG2A, MGAT1, and GET1 are currently in our pipeline. We design functional assays to test pathogenicity of variants using patient-derived cells or by functional complementation of KO lines with patient-variant constructs. Pathogenic variants do not complement, showing they are legitimate CDGs. Assays include intracellular protein trafficking, Golgi morphology and dynamics, glycomic analysis and lectin binding. The clinical discoveries enhance our insights into basic science and sometimes provide therapy for patients.