Grants and Contracts Details
Role of RFT1 in DLO assembly in human fibroblasts Protein N-glycosylation is the most common co-translational modification in the eukaryotic proteome, and is essential for the synthesis, folding, function and intracellular trafficking of eukaryotic glycoproteins. Defects in glycosylation have been identified as the underlying cause of a growing list of inherited genetic defects in humans, referred to as Congenital Disorders of Glycosylation (CDG). Asn-glycosylation requires the synthesis of a precusor oligosaccharide, containing 3 glucose (Glc), 9 mannose (Man) and 2 N-acetyl glucosamine (GlcNAc) residues, attached to a long-chain polyisoprenyl alcohol (dolichol) via a diphosphate bridge (Glc3Man9GlcNAc2-P-P-Dol, G3-DLO). The assembly of this dolichol-linked oligosaccharide (DLO) requires the sequential activity of at least 13 glycosyltransferases, several accessory polypeptides of unknown function and additional transport proteins (flippases) that have not yet been described. A distinguishing feature of the DLO assembly pathway is that synthesis is initiated on the cytoplasmic monolayer of the ER, with the formation of a Man5GlcNAc2-P-P-Dol (M5-DLO) intermediate, which is then ‘flipped’ into the lumenal compartment of the ER, where DLO synthesis is completed by the addition of 4 mannose and 3 glucose units, donated from Man-P-Dol and Glc-P-Dol (also formed in the cytosolic leaflet), to form the mature DLO, G3-DLO. Currently, mutations resulting in human CDGs (Type 1), due to deficient DLO synthesis, have been identified in at least 18 separate genes involved in either GDP-Man synthesis, dolichol phosphate synthesis, or DLO assembly. Mutations in one of these genes, RFT1, results in the accumulation of the Man5GlcNAc2-P-P-Dol intermediate and inefficient protein N-glycosylation. RFT1 was initially proposed to be the flippase protein which catalyzes the transmembrane diffusion of the M5-DLO intermediate from the cytosolic to the lumenal monolayer. Although the RFT1 protein is clearly required for efficient conversion of M5-DLO to mature DLO, several molecular and biochemical studies have challenged its proposed role as the flippase. It is plausible that RFT1 might function as a chaperone to receive the M5-DLO on the lumenal monolayer of the ER and deliver it to Alg3, the next Man-P-Dol: DLO mannosyltransferase in the pathway, or as a component of a complex of lumenal enzymes that accomplish the completion of mature DLO. Alternatively, it may have an unrelated activity which results, pleitropically, in mislocalization of M5-DLO, preventing productive interaction with the flippase complex and the DLO assembly pathway. The objective of this application
|Effective start/end date||4/1/15 → 3/31/17|
Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.