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ARS Home » Midwest Area » West Lafayette, Indiana » Crop Production and Pest Control Research » Research » Publications at this Location » Publication #82276


item Nam, Young-woo
item Jung, Rudolf
item Nielsen, Niels

Submitted to: Journal of Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/5/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Because soybean is an important agronomic crop in the United States, research is carried out by USDA-ARS to improve seed quality. A persistent problem has been that soybean proteins have an inferior nutritional quality due to suboptimal amounts of essential sulfur amino acids. Despite extensive research, conventional plant breeding approaches have been unable eto improve soybean nutritional quality and attention has now focused on biotechnological approaches. Successful application of a biotechnological approach requires detailed knowledge about the structure of the molecules targeted for modification and the mechanisms involved in the synthesis and assembly of those molecules. To facilitate the study of glycinin, the most prevalent protein in most soybeans, a procedure has been developed to synthesize glycinin subunits in a cell free system and assemble them into trimer complexes like those found in the seed. The research described in this paper discriminate between a source of chemical energy that is required for synthesis of glycinin subunits, and one causes the molecule to fold into a conformation required for assembly of trimers. The energy requirement, together with the demonstration that certain chemicals inhibit this process, implicate molecular chaperones as a catalyst required in the folding process. Molecular chaperones are proteins that assist in the correct folding of other proteins. The information in this paper provides new insight about the pathway followed by seed proteins during their maturation and will be useful to scientists working to improve the quality of grain legumes.

Technical Abstract: Seed protein proglobulins were synthesized from cDNAs in reticulocyte lysates. Most proglobulins were recovered as trimers when translation rates were low, but primarily monomers were recovered at high translation. Prevalence of monomers was accompanied by elevated amounts of insoluble protein recovered at the bottom of sucrose density gradients. Apyrase treatment of translation mixtures after synthesis, but before significant assembly occurred, drastically reduced trimer assembly and increased the proportion of insoluble aggregate. These observations indicated ATP is required for protein folding and/or trimer assembly. The appearance of insoluble aggregated protein when rates of synthesis were elevated, or when ATP was absent, suggested that protein misfolding occurred. Trimer assembly was stimulated when wheat germ translation mixtures defective in supporting efficient trimer assembly were supplemented with fractions isolated from endoplasmic reticulum of developing pea seeds. Molecular chaperones are likely involved in folding and/or assembly of proglobulin trimers both in reticulocyte lysates and in seeds. Consistent with this hypothesis, trimer formation was reduced when carboxymethylated bovine albumin and alpha-casein, considered to mimic proteins with extended chain and molten-globular conformations and thereby compete for Hsp70 and Hsp60-type molecular chaperones, respectively, were introduced into translation mixtures.