Analysis in vivo of GRP78-BiP/substrate interactions and their role in induction of the GRP78-BiP gene.

Abstract

The endoplasmic reticulum (ER)-localized chaperone protein, GRP78-BiP, is involved in the folding and oligomerization of secreted and membrane proteins, including the simian virus 5 hemagglutinin-neuraminidase (HN) glycoprotein. To understand this interaction better, we have constructed a series of HN mutants in which specific portions of the extracytoplasmic domain have been deleted. Analysis of these mutant polypeptides expressed in CV-1 cells have indicated that GRP78-BiP binds to selective sequences in HN and that there exists more than a single site of interaction. Mutant polypeptides have been characterized that are competent and incompetent for association with GRP78-BiP. These mutants have been used to show that the induction of GRP78-BiP synthesis due to the presence of nonnative protein molecules in the ER is dependent on GRP78-BiP complex formation with its substrates. These studies have implications for the function of the GRP78-BiP protein and the mechanism by which the gene is regulated. INTRODUCTIONThe 78-kDa glucose-regulated/Ig heavy-chain binding protein (GRP78-BiP) is a member of the highly con-

Stable Binding of ATF6 to BiP in the Endoplasmic Reticulum Stress Response

“…Next we sought to test a substrate that is known to cycle on and off BiP. We used the SV5 HN protein, which binds BiP transiently as part of the HN protein maturation process (28). As with ATF6, the cells were pulse-labeled with [ 35 S]methionine and immunoprecipitated with either anti-HN sera or anti-hamster BiP sera.…”

The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum.

“…Thus, neither the massive accumulation of the insoluble PiZ variant of human a1-antitrypsin nor the distention of the ER that accompanies this accumulation is sufficient to elicit increased synthesis of BiP (18). Furthermore, the accumulation in the ER of deletion mutants of the simian virus 5 hemagglutinin-neuraminidase glycoprotein that do not bind to BiP does not cause induction of the unfolded protein response (41). Finally, stimuli other than the presence of unfolded proteins in the ER may also lead to induction of BiP, since in the case of one sec mutant, secl8 (13,20), in which BiP is induced at the nonpermissive temperature, secretory proteins that accumulate in the ER appear to be properly folded (14).…”

Dissociation of glucose-regulated protein Grp78 and Grp78-IgE Fc complexes by ATP.

“…In mammalian cells, Grp78 is not induced by heat (1); however, treatments increasing the amount of malfolded proteins in the ER stimulate a dramatic increase in its synthesis (2,3). For instance, inhibitors of N-linked glycosylation induce Grp78 synthesis and this induction is correlated with an increased binding of Grp78 to underglycosylated proteins (4,5).…”