Plant Cell Advance Online Publication
Published on September 24, 2003; 10.1105/tpc.013052
Received April 17, 2003
Accepted July 15, 2003
A Phaseolin Domain Involved Directly in Trimer Assembly Is a Determinant for Binding
by the Chaperone BiP
Ombretta Foresti 1, Lorenzo Frigerio 2, Heidi Holkeri 1, Maddalena de Virgilio 1, Stefano Vavassori 1, and Alessandro Vitale 1*
1
Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche,
20133 Milano, Italy
2
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United
Kingdom
* To whom correspondence should be addressed. E-mail: vitale{at}ibba.cnr.it.
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.
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