Role of Individual Disulfide Bonds in the Structural Maturation of a Low Molecular Weight Glutenin Subunit

Andrea Orsi,Francesca Sparvoli,Aldo Ceriotti

doi:10.1074/jbc.m103833200

Abstract

Gliadins and glutenins are the major storage proteins that accumulate in wheat endosperm cells during seed development. Although gliadins are mainly monomeric, glutenins consist of very large disulfide-linked polymers made up of high molecular weight and low molecular weight subunits. These polymers are among the largest protein molecules known in nature and are the most important determinants of the viscoelastic properties of gluten. As a first step toward the elucidation of the folding and assembly pathways that lead to glutenin polymer formation, we have exploited an in vitro system composed of wheat germ extract and bean microsomes to examine the role of disulfide bonds in the structural maturation of a low molecular weight glutenin subunit. When conditions allowing the formation of disulfide bonds were established, the in vitro synthesized low molecular weight glutenin subunit was recovered in monomeric form containing intrachain disulfide bonds. Conversely, synthesis under conditions that did not favor the formation of disulfide bonds led to the production of large aggregates from which the polypeptides could not be rescued by the post-translational generation of a more oxidizing environment. These results indicate that disulfide bond formation is essential for the conformational maturation of the low molecular weight glutenin subunit and suggest that early folding steps may play an important role in this process, allowing the timely pairing of critical cysteine residues. To determine which cysteines were important to maintain the protein in monomeric form, we prepared a set of mutants containing selected cysteine to serine substitutions. Our results show that two conserved cysteine residues form a critical disulfide bond that is essential in preventing the exposure of adhesive domains and the consequent formation of aberrant aggregates.

Highlights

Gliadins and glutenins are the major storage proteins that accumulate in wheat endosperm cells and are largely responsible for the unique suitability of wheat flour for bread-making
The detailed structure of glutenin polymers remains unknown, available data indicate that low molecular weight (LMW) glutenin subunits are linked via disulfide bonds to other subunits of the same class and to high molecular weight (HMW) glutenin subunits and to polypeptides related to ␥-gliadins [2]
Our results suggest that formation of soluble monomeric subunits is an early step on the pathway of glutenin polymer assembly and indicate that one intrachain disulfide bond plays a major role in monomer maturation, possibly by maintaining adhesive domains in a buried state and, preventing the precocious aggregation of the newly synthesized polypeptides

Summary

EXPERIMENTAL PROCEDURES

Recombinant DNA Techniques—Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer’s instructions. For separation under reducing conditions, samples were mixed with 6 volumes of 200 mM Tris-HCl, pH 8.6, 6% SDS, 8% glycerol, 50 mM DTT, 0.01% bromphenol blue and heated for 5 min at 100 °C. Sedimentation Velocity Analysis on Sucrose Gradients—For sedimentation velocity analysis on sucrose gradients, in vitro translations were terminated by adding cycloheximide at a final concentration of 2 mM. N-Ethylmaleimide was added at a final concentration of 40 mM, and microsomes were recovered by centrifugation at 35,000 rpm for 10 min at 4 °C in a SW 55 Ti rotor (Beckman Instruments) through a high salt/sucrose cushion (250 mM sucrose, 500 mM potassium acetate, 5 mM magnesium acetate, 50 mM Hepes-KOH, pH 7.9) [30]. Equivalent amounts of gradient fractions and resuspended pellet were diluted with half a volume of 600 mM Tris-HCl, pH 8.6, 25%

In Vitro Folding of a LMW Glutenin Subunit

RESULTS

DISCUSSION