Intracellular fate of fibrinogen B beta chain expressed in COS cells.
Full-length fibrinogen B beta cDNA was subcloned into an expression vector, pBC12BI, and transfected into COS cells. B beta chain expression was measured by pulse-labelling cells with L-[35S]methionine, immunoprecipitating the B beta chain with antibody to fibrinogen and separating the nascent radioactive protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). B beta chain was expressed in transfected COS cells but was not secreted into the medium. Treatment with endoglycosidase H showed that non-secreted B beta chain contains mannose-rich carbohydrates rather than the complex form of carbohydrate which occurs in plasma fibrinogen and indicates that B beta chain is not transported to the Golgi apparatus. In transfected COS cells, antibody to fibrinogen co-immunoprecipitated B beta chain and 78 kDa immunoglobulin-binding protein (BiP) and antibody to BiP immunoprecipitated BiP and nascent B beta chains. Non-secreted B beta chain was degraded intracellularly with a half-life of 5 h by enzymes which were not affected by incubating transfected cells with NH4Cl, which indicates a non-lysosomal pathway of degradation. These studies indicate that B beta chain by itself does not contain the signal for fibrinogen secretion and that non-secreted B beta chain is associated with BiP and degraded in the rough endoplasmic reticulum.
Danishefsky
K
K
Lindsley F. Kimball Research Institute, New York Blood Center, NY 100221.
Hartwig
R
R
Banerjee
D
D
Redman
C
C
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D005340:Fibrinogen / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D015183:Restriction Mapping; D014162:Transfection
D046911:Macromolecular Substances; D005340:Fibrinogen