A Relaxed Specificity in Interchain Disulfide Bond Formation Characterizes the Assembly of a Low-Molecular-Weight Glutenin Subunit in the Endoplasmic Reticulum    

Abstract

Wheat (Triticum spp.) grains contain large protein polymers constituted by two main classes of polypeptides: the highmolecular-weight glutenin subunits and the low-molecular-weight glutenin subunits (LMW-GS). These polymers are among the largest protein molecules known in nature and are the main determinants of the superior technological properties of wheat flours. However, little is known about the mechanisms controlling the assembly of the different subunits and the way they are arranged in the final polymer. Here, we have addressed these issues by analyzing the formation of interchain disulfide bonds between identical and different LMW-GS and by studying the assembly of mutants lacking individual intrachain disulfides. Our results indicate that individual cysteine residues that remain available for disulfide bond formation in the folded monomer can form interchain disulfide bonds with a variety of different cysteine residues present in a companion subunit. These results imply that the coordinated expression of many different LMW-GS in wheat endosperm cells can potentially lead to the formation of a large set of distinct polymeric structures, in which subunits can be arranged in different configurations. In addition, we show that not all intrachain disulfide bonds are necessary for the generation of an assembly-competent structure and that the retention of a LMW-GS in the early secretory pathway is not dependent on polymer formation.

Redox regulation of glutenin subunit assembly in the plant endoplasmic reticulum

“…Previously, assembly of some high‐molecular‐weight glutenin subunits was studied in transgenic tobacco (Robert et al ., ; Shani et al ., ), and polymerization of a LMW‐GS from Triticum aestivum cv. Cheyenne, known as B11‐33, has also been investigated by utilizing transient expression in tobacco protoplasts (Lombardi et al ., ). Inter‐chain disulfide bonds were shown to be crucial for B11‐33 polymer formation.…”

“…Inter‐chain disulfide bonds were shown to be crucial for B11‐33 polymer formation. However, rather than being governed by specific pairing between subunits, assembly appears to rely on a relaxed specificity with regard to disulfide bond formation, whereby each cysteine residue may form a disulfide bond with a variety of companion cysteines present on identical or different subunits (Lombardi et al ., ). This would drive the formation of a large number of complex glutenin polymers.…”

Characterization of durum wheat high molecular weight glutenin subunits Bx20 and By20 sequences by a molecular and proteomic approach

“…Therefore, the presence of two cysteines does not seem to be a sufficient condition to influence positively the glutenin polymer growth, which is based on HMW‐GS x‐ and y‐type interactions, forming the pillar of glutenin polymers. As a further support to the hypothesis of the role of the number and position of cysteine residues, Lombardi et al demonstrated that glutenin polymer formation was affected by the mutation of specific cysteine residues in low molecular weight glutenin subunits.…”

Trafficking and deposition of prolamins in wheat

“…The mechanisms regulating the folding and assembly of wheat gliadins and LMW glutenin subunits have been studied in heterologous in vitro systems by several groups Altschuler and Galili, 1994;Lombardi et al, 2009;Rosenberg et al, 1993). While the results of these studies seem to suggest a role for interactions of BiP and PDI in the processing of these prolamins, the exact nature of these interactions remains poorly understood.…”

“…However, more recent studies (Francin-Allami et al, 2011) carried out in tobacco cells have shown that tandem repeats are not the only sequences involved in ER retention of g-gliadin since both the isolated repetitive N-terminal and the cysteine-rich C-terminal domains of the protein were able to be retained and accumulated as protein body-like structures. Structural features within a LMW-GS have been suggested to determine the retention of this protein in the ER of tobacco protoplasts (Lombardi et al, 2009), while its non-repetitive Cterminal domain, in particular, has been implicated in its retention in Xenopus oocytes (Altschuler and Galili, 1994).…”