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.