A Phaseolin Domain Involved Directly in Trimer Assembly Is a Determinant for Binding by the Chaperone BiP

Abstract

The binding protein (BiP; a member of the heat-shock 70 family) is a major chaperone of the endoplasmic reticulum (ER). Interactions with BiP are believed to inhibit unproductive aggregation of newly synthesized secretory proteins during folding and assembly. In vitro, BiP has a preference for peptide sequences enriched in hydrophobic amino acids, which are expected to be exposed only in folding and assembly intermediates or in defective proteins. However, direct information regarding sequences recognized in vivo by BiP on real proteins is very limited. We have shown previously that newly synthesized monomers of the homotrimeric storage protein phaseolin associate with BiP and that phaseolin trimerization in the ER abolishes such interactions. Using different phaseolin constructs and green fluorescent protein (GFP) fusion proteins, we show here that one of the two ␣ -helical regions of polypeptide contact in phaseolin trimers (35 amino acids located close to the C terminus and containing three potential BiP binding sites) effectively promotes BiP association with phaseolin and with secretory GFP fusions expressed in transgenic tobacco or in transfected protoplasts. We also show that overexpressed BiP transiently sequesters phaseolin polypeptides. We conclude that one of the regions of monomer contact is a BiP binding determinant and suggest that during the synthesis of phaseolin, the association with BiP and trimer formation are competing events. Finally, we show that the other, internal region of contact between monomers is necessary for phaseolin assembly in vivo and contains one potential BiP binding site.

Endoplasmic Reticulum Protein Quality Control and Its Relationship to Environmental Stress Responses in Plants

“…The binding of BiP to proteins is released by ATP binding through nucleotide exchange (Wei et al, 1995); therefore, cycles of nucleotide hydrolysis and exchange drive the binding and release BiP from unfolded or misfolded protein substrates, a process that terminates when the hydrophobic sequences in the protein substrate are buried (Gething, 1999). One of the best examples of the role of BiP in the biogenesis of a secreted protein is the binding of BiP to sites in monomers of the trimeric bean protein Phaseolin that are buried in the mature protein (Vitale et al, 1995;Foresti et al, 2003). The binding and release cycles are regulated by cofactors, such as DNAJ proteins that promote ATP hydrolysis or ATP:ADP exchange (Cheetham and Caplan, 1998).…”

Zeolin. A New Recombinant Storage Protein Constructed Using Maize γ-Zein and Bean Phaseolin

“…On the whole, our results suggest that BiP directly interacts with the zein portion of zeolin, but this remains to be demonstrated. The C-terminal a-helical region of phaseolin, which is involved in trimerization, is a BiP-binding determinant in phaseolin monomers (Foresti et al, 2003), but we do not know if it is still available for BiP interactions in disulfide-bonded, insoluble zeolin oligomers. Finally, it should be noted that phaseolin-KDEL, which is also efficiently retained in the ER, does not interact with BiP more extensively than wild-type phaseolin (Frigerio et al, 2001), indicating that residence in the ER, per se, is not sufficient to create a strong BiP ligand.…”

“…For immunoprecipitation in reducing conditions, the immunoprecipitation buffer was supplemented with 4% 2-mercaptoethanol. ATP-mediated release of BiP from immunoprecipitates was performed as described by Foresti et al (2003). Analysis of immunoprecipitated material was by SDS-PAGE followed by fluorography.…”

Endoplasmic Reticulum Stress Response

“…Soybean BiP has also been shown to associate detectably with normal storage proteins in vitro . However, direct evidence for a role of plant BiP in storage protein folding and maturation includes the demonstration that BiP associates co-translationally with rice prolamin storage proteins (Li et al, 1993) and binds to exposed sites on monomers of phaseolin but not to the trimeric form of the bean protein (Foresti et al, 2003). Thus, the BiP binding site is buried in the mature bean protein, supporting the notion that BiP action precedes and contributes to the maturation of legume storage proteins.…”