Abstract
Wheat (Triticum spp.) grains contain large protein polymers constituted by two main classes of polypeptides: the high-molecular-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.
Highlights
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)
The C-terminal part of the protein consists of a domain that is stabilized by the presence of three intrachain disulfide bonds and that can be further subdivided into three regions (I, II, and III in Fig. 1): a first region containing 5 Cys residues, Cc, Cd, Ce, Cf1, and Cf2, all involved in the formation of intrachain disulfides; a Gln-rich domain containing a Cys residue (Cx) that remains available for polymer formation; and a C-terminal conserved sequence containing one further Cys residue (Cy) that is again involved in the formation of an intrachain bond
When FLAG-tagged forms of the two B1133 Cys mutants were individually coexpressed with hemagglutinin epitope (HA)-tagged mutants of the 1B protein, a band of the expected size could be invariably immunoselected by both the anti-FLAG and anti-HA antibodies (Supplemental Fig. S1). This confirmed the formation of four different types of covalent heterodimers between the two proteins. These results indicate that 1B and B1133 proteins can form mixed disulfides involving any possible combination of the two Cys residues that remain available for interchain disulfide bond formation in the folded monomers
Summary
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). 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. Of a Low-Molecular-Weight Glutenin Subunit which the majority of B-type subunits belong, would act as chain extenders, because they contain two Cys residues that remain available for the formation of interchain disulfide bonds. Our results confirm that disulfide bonds are crucial for the assembly of these proteins and indicate that a relaxed specificity in Cys pairing from different subunits can drive the formation of complex glutenin polymers
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