ARRA: Regulation of Protein N-Glycosylation in the CNS

  • Waechter, Charles (PI)

Grants and Contracts Details


The long-term goal of this project continues to be to elucidate all of the regulatory controls imposed on the biosynthesis and transbilayer movement of dolichol-linked saccharide intermediates involved in the co- translational N-glycosylation of secretory and membrane glycoproteins in mammalian cells. One of the Specific Aims of the previous proposal was to determine of the glycosyl carrier lipid was "recycled" to the cytoplasmic face of the ER as dolichyl phosphate (Dol-P) or the free polyisoprenol, dolichol. The results of the studies outlined in the Experimental Procedures provided strong evidence that the Do]-P-P discharged when the precursor oligosaccharide was transferred to nascent polypeptide acceptors during the primary N-glycosylation event the ER lumen, it was rapidly cleaved to Dol-P. Additional studies have now established that this pool of dol-P is "recycled" to the cytoplasmic surface to reutilized for additional rounds of lipid intermediate biosynthesis. Since biophysical studies indicate that Dol-P does not "flip-flop" in an unassisted process, this results suggests that there is an ER protein(s) that functions as a novel, and unexpected, Dol-P flippase to facilitate the transbilayer movement of the carrier lipid. When the initial proposal was written, it was assumed that Dol-P would be dephosphorylated and free dolichol could diffuse more readily without the need for a flippase. The free polyisoprenol could have been rephosphorylated on the cytoplasmic face by dolcihol kinase, and then be available for new rounds of lipid intermediate biosynthesis. Preliminary evidence from an analogue- based transport assay has strengthened the evidence for this novel flippase, and the effects of the CWHS mutation in S. cerevisiae indicate that recycling of the carrier lipid after Dol-P-P is released on the lumenal surface contributes significantly to the pool of Dol-P on the cytoplasmic monolayer.
Effective start/end date9/30/098/31/11


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