In vitro synthesis and assembly of legume seed storage globulins
Abstract
Globulins are the major storage proteins of legume seeds. They are synthesized and assembled during a complex process involving both post-translational modification and subcellular transport in developing cotyledon cells. Assembly of proglobulin trimers in vitvo in a reticulocyte lysate system requires ATP, which suggests that folding and assembly of globulin subunits may be assisted by molecular chaperones. Addition of unfolded proteins able to compete for chaperones to this cell-free system prevents trimer assembly. Similar assembly experiments in a wheat germ extract system were not successful unless extracts from endoplasmic reticulum isolated from developing pea cotyledons were added. These observations strongly implicate the participation of molecular chaperones in the assembly of seed storage globulins in vivo. Assembly of 11S proglobulin subunits into trimers and hexamers in vitro also depends on an oxidized redox state of glutathione in the assembly mixture. This effect was not observed with a cysteine mutant of the prolegumin-B subunit that lacked the interchain disulfide bridge between acidic and basic polypeptides. Proteolytic processing and reassembly of the cysteine mutant demonstrated that it was incapable of assembly into hexamers despite specific cleavage into acidic and basic polypeptides with asparaginyl endopeptidase. Thus, the interchain disulfide bridge is crucial for maintaining the structure of 11S globulin trimers during assembly into hexamers. Heterogeneous trimers formed between wild type and the cysteine mutant subunits were tested for hexamer assembly. Unlike the trimers composed of only mutant subunits, subunits of the mixed trimers assembled into hexamers. This observation supported in vivo data that showed the cysteine mutants could accumulate as mature hexamers in protein bodies of transgenic tobacco seeds. To investigate possible interactions between 11S and 7S globulins, bacterially expressed prolegumin subunits were denatured and permitted to fold and self-assemble with in vitro synthesized $\beta$-conglycinin subunits. The two proteins did not interact to form any heterogeneous trimers. Reassembly of dissociated native 11S globulins in the presence of in vitro synthesized $\beta$-conglycinin subunits also demonstrated an inability to form heterogeneous hexamers.
Degree
Ph.D.
Advisors
Nielsen, Purdue University.
Subject Area
Agronomy|Biochemistry|Molecular biology
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