Grants and Contracts Details
Description
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.
Status | Finished |
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Effective start/end date | 9/30/09 → 8/31/11 |
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