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ARS Home » Midwest Area » West Lafayette, Indiana » Crop Production and Pest Control Research » Research » Publications at this Location » Publication #82270
Title: PROCESSING AND ASSEMBLY OF 11S SEED STORAGE GLOBULINS IN VITRO AND IN TRANSGENIC TOBACCO SEEDS: SPECIFICITY OF LIMITED PROTEOLYSIS AND ITS ROLE IN 11S GLOBULIN ASSEMBLY

Author
item JUNG, RUDOLF - PURDUE UNIV & IPK
item SCOTT, M - PURDUE UNIVERSITY
item NAM, YOUNG-WOO - PURDUE UNIVERSITY
item BEAMAN, TODD - PURDUE UNIVERSITY
item BASSUNER, RONALD - PURDUE UNIVERSITY
item SAALBACH, ISOLDE - IPK INST FUR PFLAM & KULT
item MUNTZ, KLAUS - IPK INST FUR PFLAM & KULT
item NIELSEN, NIELS

Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/17/1997
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 to improve soybean nutritional quality and attention is now focused on biotechnological approaches. Successful application of such an approach requires detailed knowledge both about the structure of the molecules targeted for modification and the mechanisms involved in the synthesis and assembly of the target 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 describe the substrate requirements of an enzyme required to convert subunit precursors to a mature form. It shows that it is necessary to cut subunits in glycinin trimers before they can assemble into hexamers, a necessary step in the maturation process that takes place in the seed. The information in this paper provides new insight about the pathway followed during the maturation of glycinin storage proteins and will be useful to scientists working to improve the quality of grain legumes.

Technical Abstract: The 11S seed storage proteins are synthesized as precursors and cleaved post-translationally by an asparaginyl endopeptidase. To study the specificity of reactions catalyzed by this asparaginyl endopeptidase, a series of octapeptides and either mutant legumin-B or G4 glycinin subunits were prepared that contained amino acid mutations in the region surrounding gthe conserved cleavage site. The endopeptidase had an absolute specificit for Asn on the amino-terminal side of the peptide bond that was cleaved but exhibited little specificity for amino acids on the carboxyl-terminal side. The sequence conservation observed immediately to the carboxyl-terminal side of the cleavage site among the many 11S seed storage globulins whose primary structures are known is therefore not a feature directly related to post-translational modification. Rather, the specificity is likely related to a conservation of structure around the highly conserved cysteine residue einvolved in the formation of the highly conserved disulfide bond that link the acidic and basic chains of the mature subunit. To evaluate the function of post-translational cleavage, the capacity of unmodified and modified subunits to assemble into hexamers equivalent to those isolated from seeds was tested. The results showed cleavage of proglobulin subunits in trimers is required for their assembly into hexamers in vitro. Moreover, products from a mutant gene encoding a non-cleavable prolegumin subunit were accumulated as trimers rather than hexamers in seeds of transgenic tobacco, whereas products from the unmodified cleavable prolegumin B gene were accumulated as hexamers. These data clearly establish that 11S proglobulins must be modified post-translationally for normal assembly into hexamers to occur in protein storage vacuoles.