Epitope Tagging of the Human Endoplasmic Reticulum HSP70 Protein, BiP, to Facilitate Analysis of BiP-Substrate Interactions

doi:10.1006/abio.1995.1399

Analytical Biochemistry

Volume 229, Issue 2, August 1995, Pages 170-179

https://doi.org/10.1006/abio.1995.1399 Get rights and content

Abstract

We modified BiP, the resident endoplasmic reticulum (ER) heat shock protein 70, to contain an epitope-tag 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.

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Citation Excerpt :
To determine if the other subunits associate with BiP, we performed a similar set of experiments characterizing the association of BiP with AChR subunits as with ERp57 (Fig. 3). Again, we used an HA-tagged construct (BiP-HA) (41) to look at associations between BiP and AChR subunits because of problems quantitatively precipitating endogenous BiP (data not shown). In contrast to ERp57-HA, transfection of BiP-HA resulted in only a ∼35% increase (n = 4) of BiP expression (Fig. 3A).
We examined interactions between the endoplasmic reticulum (ER) chaperones calnexin (CN), ERp57, and immunological heavy chain-binding protein (BiP) and nicotinic acetylcholine receptor (nAChR) subunits. The three chaperones rapidly associate with newly synthesized nAChR subunits. Interactions between nAChR subunits and ERp57 occur via transient intermolecular disulfide bonds and do not require subunit N-linked glycosylation. The associations of ERp57 or CN with AChR subunits are long lived and prolong subunit lifetime ∼10-fold. Coexpression of CN or ERp57 alone does not affect nAChR assembly or trafficking, but together they cause a significant decrease in nAChR expression and assembly. In contrast, associations with BiP are shorter lived and do not alter nAChR expression and assembly. However, a mutated BiP that slows its dissociation significantly increases its associations and decreases nAChR expression and assembly. Our results suggest that interactions with the chaperones regulate the levels of nAChRs assembled in the ER by stabilizing and sequestering subunits during assembly.
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Regular ArticleEpitope Tagging of the Human Endoplasmic Reticulum HSP70 Protein, BiP, to Facilitate Analysis of BiP-Substrate Interactions

Abstract

Regular Article
Epitope Tagging of the Human Endoplasmic Reticulum HSP70 Protein, BiP, to Facilitate Analysis of BiP-Substrate Interactions