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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
Author(s) -
Feige JeanJacques,
Keller GilbertA.,
Scheffler Immo E.
Publication year - 1988
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041360105
Subject(s) - endoplasmic reticulum , endoglycosidase h , chinese hamster ovary cell , glycoprotein , receptor , mannose , mutant , transfection , glycosylation , biology , microbiology and biotechnology , hamster , protein biosynthesis , biochemistry , golgi apparatus , gene
Abstract 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 40deg;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 40deg;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.