Temperature-sensitive Chinese hamster cell mutant with a defect in glycoprotein synthesis: accumulation of the EGF receptor in the endoplasmic reticulum and the role of the glucose-regulated protein GRP78.
A temperature-sensitive mutant of Chinese hamster fibroblasts with a defect in glycoprotein synthesis is investigated after transfection and amplification of the gene for the human EGF receptor. We demonstrate that at the nonpermissive temperature a partially glycosylated species of the receptor accumulates in the endoplasmic reticulum. The oligosaccharides present are the high mannose types, since they can be removed completely by treatment with endoglycosidase H. Pulse-chase experiments show that the abnormal species of the receptor cannot be chased to a form that is either resistant to endoglycosidase H, or altered in its mobility on SDS polyacrylamide gels. The abnormal species of the receptor appears within the first hour of a shift to the nonpermissive temperature, and no further changes are observed upon prolonged incubation of cells at 40 degrees C. However, after 3-4 hours immunoprecipitations of the receptor yield another protein, which has properties very similar, if not identical, to the glucose-regulated protein GRP78. The induction of this protein at 40 degrees C can be suppressed completely with an inhibitor of RNA synthesis, without any effect on the glycosylation defect, or on the accumulation of the EGF receptor in the endoplasmic reticulum.
Feige
J J
JJ
Department of Biology, University of California, San Diego 92093.
Keller
G A
GA
Scheffler
I E
IE
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D066246:ErbB Receptors / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000096:biosynthesis*; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D013696:Temperature
D000091342:Endoplasmic Reticulum Chaperone BiP; D006023:Glycoproteins; D018840:HSP70 Heat-Shock Proteins; C000717247:HSPA5 protein, human; D008565:Membrane Proteins; C018551:glucose-regulated proteins; D066246:ErbB Receptors