Accumulation of glucosaminyl(acyl)phosphatidylinositol in an S3 HeLa subline expressing normal dolicholphosphomannose synthase activity.
Glucosaminyl(acyl)phosphatidylinositol [GlcN(acyl)PI], the third intermediate in the mammalian glycosylphosphatidylinositol (GPI) anchor pathway, is undetectable in most cells. This intermediate was previously shown to accumulate, however, in murine lymphoma mutant E and in yeast mutant dpm1, both of which lack dolicholphosphomannose synthase activity. Here we report that a mammalian HeLa S3 subline, denoted D, produces large amounts of GlcN(acyl)PI. The level of GlcN(acyl)PI in this subline is twice that in the murine lymphoma mutant E and 4 times that in the parental S3 line. This HeLa D subline differs from the previously reported mutants that accumulate GlcN(acyl)PI because no defects in the synthesis or utilization of dolicholphosphomannose were found. Kinetic analysis indicated that in this HeLa subline there is an increased rate of synthesis of GlcN(acyl)PI, whereas the rate of metabolism for this GPI is comparable to that in wild-type cells. Furthermore, HeLa D cells accumulate GlcN(acyl)PI without a block in the synthesis of the downstream mannosylated GPI anchor precursors and GPI-anchored proteins. These findings might be relevant for understanding the regulation of the GPI pathway.
Sevlever
D
D
Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, U.S.A.
Schiemann
D
D
Guidubaldi
J
J
Medof
M E
ME
Rosenberry
T L
TL
D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D005944:Glucosamine / Q000031:analogs & derivatives*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007294:Inositol / Q000378:metabolism; D007700:Kinetics; D008362:Mannosides / Q000096:biosynthesis; D008364:Mannosyltransferases / Q000378:metabolism*; D008858:Microscopy, Phase-Contrast; D009844:Oligosaccharides / Q000096:biosynthesis; D010716:Phosphatidylinositols / Q000378:metabolism*; D011863:Radioimmunoassay
D017261:Glycosylphosphatidylinositols; D008362:Mannosides; D009844:Oligosaccharides; D010716:Phosphatidylinositols; D007294:Inositol; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D005944:Glucosamine