Epitope tagging of the human endoplasmic reticulum HSP70 protein, BiP, to facilitate analysis of BiP--substrate interactions.
We modified BiP, the resident endoplasmic reticulum (ER) heat shock protein 70, to contain an epitopetag sequence close to the C-terminus (BiP-tag); the epitope is derived from an influenza hemagglutinin (HA) subtype and is recognized by the monoclonal antibody 12CA5. This antibody both immunoprecipitates BiP-tag and detects it on Western blots. Using transient expression of cDNAs in COS cells, we studied the interaction of BiP-tag with several membrane proteins. Consistent with previous work on BiP, BiP-tag bound poorly and transiently to newly made wild-type influenza HA glycoprotein and strongly and irreversibly to an HA mutant that misfolds and is retained in the ER. Most newly made erythropoietin receptor (EPO-R) polypeptides are retained in the ER and degraded there; we show here that, in cotransfected COS cells, newly made EPO-R is bound to BiP-tag prior to its degradation. Thus, by several criteria the BiP-tag molecule is fully functional in binding newly made proteins. Because it can be immunoprecipitated by a readily available antibody, it offers several advantages to the study of protein folding in the ER and the role of chaperones in this process.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D000939:Epitopes / Q000032:analysis*; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006360:Heat-Shock Proteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral / Q000378:metabolism; D006801:Humans; D007668:Kidney; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017467:Receptors, Erythropoietin / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D014162:Transfection
D002352:Carrier Proteins; D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; D000939:Epitopes; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D018832:Molecular Chaperones; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins