Two different mutants blocked in synthesis of dolichol-phosphoryl-mannose do not add glycophospholipid anchors to membrane proteins: quantitative correction of the phenotype of a CHO cell mutant with tunicamycin.
The addition of glycophospholipid (GPL) anchors to certain membrane proteins occurs in the rough endoplasmic reticulum and is essential for transport of the proteins to the plasma membrane. Limited circumstantial evidence suggests that dolichol-phosphoryl-mannose (DPM) is a donor of mannose residues of these anchors. We here report studies of a CHO cell mutant (B421) transfected to express the GPL-anchored protein, placental alkaline phosphatase (AP). Only a few transfectants were found to express GPL-anchored AP on their surface, and these clones synthesized DPM. Moreover, and most strikingly, when surface AP-negative transfectants were treated with tunicamycin to cause accumulation of DPM, these cells expressed lipid-anchored AP. Fusion of a cloned surface AP-negative transfectant of B421 with the Thy-1-class E mutant thymoma, which is also deficient in DPM synthesis, produced hybrids that synthesized DPM and expressed AP and Thy-1. Thus, two mutations can interrupt DPM synthesis, and three sets of observations point to an essential role of DPM for addition of GPL anchors.
Singh
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Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106.
Tartakoff
A M
AM
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D002459:Cell Fusion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004287:Dolichol Monophosphate Mannose / Q000378:metabolism*; D005816:Genetic Complementation Test; D017261:Glycosylphosphatidylinositols; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009154:Mutation; D010716:Phosphatidylinositols / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D000954:Antigens, Surface; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D010716:Phosphatidylinositols; D011134:Polysaccharides; D011994:Recombinant Proteins; D014415:Tunicamycin; D004287:Dolichol Monophosphate Mannose; D000469:Alkaline Phosphatase