The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.
The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines.
Beck
P J
PJ
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Orlean
P
P
Albright
C
C
Robbins
P W
PW
Gething
M J
MJ
Sambrook
J F
JF
D000818:Animals; D002460:Cell Line; D005800:Genes, Fungal; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis; D006031:Glycosylation; D006602:Hexosyltransferases / Q000235:genetics*; D007700:Kinetics; D008364:Mannosyltransferases / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014162:Transfection
D006023:Glycoproteins; D009844:Oligosaccharides; D006602:Hexosyltransferases; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase