During evolution the average chain length of polyisoprenoid glycosyl carrier lipids increased from C55 (prokaryotes) to C75 (yeast) to C95 (mammalian cells). In this study the ability of the E. coli enzyme, undecaprenyl pyrophosphate synthase (UPPS) to complement the loss of the yeast cis-isoprenyltransferase (cis-IPTase) in the rer2Delta mutant was tested to determine if (55)dolichyl phosphate (Dol-P) could functionally substitute in the protein N-glycosylation pathway for (75)Dol-P, the normal isoprenologue synthesized in S. cerevisiae. First, expression of UPPS in the yeast mutant was found to complement the growth and the hypoglycosylation of carboxypeptidase Y (CPY) defects suggesting that the (55)polyprenyl-P-P intermediate was converted to (55)Dol-P, and that (55)Dol-P could effectively substitute for (75)Dol-P in the biosynthesis and function of Man-P-Dol, Glc-P-Dol and Glc(3)Man(9)GlcNAc(2)-P-P-Dol (mature DLO) in the protein N-glycosylation pathway and GPI anchor assembly. In support of this conclusion mutant cells expressing UPPS: 1) synthesized (55)Dol-P based on MS analysis; 2) utilized (55)Dol-P to form Man-P-(55)Dol in vitro and in vivo and 3) synthesized N-linked glycoproteins at virtually normal rates as assessed by metabolic labeling with [(3)H]mannose. In addition, an N-terminal GFP-tagged construct of UPPS was shown to localize to the ER of CHO cells. Consistent with the synthesis of (55)Dol-P by the transfected cells, microsomes from the transfected cells synthesized the [(14)C](55)polyprenyl-P-P intermediate when incubated with [(14)C]isopentenyl pyrophosphate (I-P-P), and [(3)H]Man-P-(55)Dol when incubated with GDP-[(3)H]Man. These results indicate that (C55)polyisoprenoid chains, significantly shorter than the natural glycosyl carrier lipid, can function in the transbilayer movement of DLOs in the ER of yeast and mammalian cells, and that conserved sequences in the cis-IPTases are recognized by, yet to be identified, binding partners in the ER of mammalian cells.
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|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
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