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 pr otein 70, to contain an epitope-tag sequence close to the C-terminus ( Hip-tag); the epitope is derived from an influenza hemagglutinin (HA) subtype and is recognized by the monoclonal antibody 12CA5. This antib ody both immunoprecipitates BiP-tag and detects it on Western blots. U sing transient expression of cDNAs in COS cells, we studied the intera ction of BiP-tag with several membrane proteins. Consistent with previ ous work on Hip, BiP-tag bound poorly and transiently to newly made wi ld-type influenza HA glycoprotein and strongly and irreversibly to an HA mutant that misfolds and is retained in the ER. Most newly made ery thropoietin receptor (EPO-R) polypeptides are retained in the ER and d egraded there; we show here that, in cotransfected COS cells, newly ma de EPO-R is bound to BiP-tag prior to its degradation. Thus, by severa l criteria the BiP-tag molecule is fully functional in binding newly m ade proteins. Because it can be immunoprecipitated by a readily availa ble antibody, it offers several advantages to the study of protein fol ding in the ER and the role of chaperones in this process. (C) 1995 Ac ademic Press, Inc.