Isolation of Chinese hamster ovary cell lines temperature conditional for the cell-surface expression of integral membrane glycoproteins.
A procedure is described to select mutants of Chinese hamster ovary cells that are conditionally defective for the cell-surface expression of integral membrane glycoproteins, including the hemagglutinin (HA) of influenza virus. Using a combination of cell sorting and biochemical screening, seven cell lines were obtained that express more cell-surface HA at 32 degrees C than at 39 degrees C. The production of infectious vesicular stomatitis virus, whose growth requires insertion of an integral membrane protein into the plasma membrane, was also temperature conditional in the majority of these mutant cell lines. Five of the lines synthesized apparently normally core-glycosylated HA at the elevated temperature but the protein was neither displayed on the cell surface nor accumulated intracellularly. In these cell lines, little or no terminally glycosylated HA molecules were observed after synthesis at 39 degrees C. By contrast, the core glycosylation of HA and several other integral membrane proteins was abnormal in the remaining two cell lines at both permissive and restrictive temperatures, due to a lesion in a cellular gene(s) that affects the formation of and/or the addition of mannose-rich oligosaccharide chains to newly synthesized polypeptides. Although HA was transported to the plasma membrane at both 32 and 39 degrees C, it did not accumulate on the cell surface at the higher temperature, apparently because of an increased rate of degradation.
Hearing
J
J
Department of Microbiology, State University of New York, Stony Brook 11794.
Hunter
E
E
Rodgers
L
L
Gething
M J
MJ
Sambrook
J
J
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002999:Clone Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000378:metabolism; D007163:Immunosorbent Techniques; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010053:Ovary; D013696:Temperature; D014721:Vesicular stomatitis Indiana virus / Q000254:growth & development
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D008562:Membrane Glycoproteins