Synthesis and assembly of soybean ?-conglycinin in vitro

Abstract

The construction of SP6-derived expression plasmids that encode normal and modified beta-conglycinin subunits is described. With the exception of an additional methionine at their NH2-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids corresponded to mature alpha and beta subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH2-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal alpha subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified beta-conglycinin subunits. The results also show that structural changes at the NH2-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.

Structure and characterization of the gene encoding .ALPHA. subunit of soybean .BETA.-conglycinin.

“…The organization is similar to the genes for other subunits (α' and β) of β-conglycinin and to the genes for legume 7S globulin type proteins, in that numbers and positions of introns are similarly organized (Doyle et al, 1986;Higgins et al, 1988;Harada et al, 1989;Takei et al, 1989). The deduced amino acid sequence was identical to that reported for the cognate cDNA clone (Lelievre et al, 1992). This fact, together with the exonintron organization of the gene, indicates that the genomic clone described here actually encodes the a subunit mRNA.…”

“…Nucleotide (top) and the deduced amino acid sequences (bottom) of the β-conglycinin α subunit gene. Translation start (ATG) and stop (TGA) codons, and poly(A)-addition-site as determined from the corresponding cDNA sequence (Lelievre et al, 1992) are boxed. Introns 1 to 5 are underlined.…”

Accumulation of β-Conglycinin in Soybean Cotyledon through the Formation of Disulfide Bonds between α′- and α-Subunits

“…The b-conglycinin trimers are mixtures of isoforms consisting of different combinations of the three types of subunits, designated a#, a with M r of 57,000, and b with M r of 42,000 (Thanh and Shibasaki, 1978;Davies et al, 1985;Nielsen and Nam, 1999). The a#-, a-, and b-subunits are synthesized in the endoplasmic reticulum (ER) of the cotyledon cell as polypeptides with a signal peptide and a propeptide (prepro a#-and prepro a-subunits) or as polypeptides with only a signal peptide (pre-b-subunit; Sengupta et al, 1981;Coates et al, 1985;Harada et al, 1989;Sebastiani et al, 1990;Lelievre et al, 1992). Each signal peptide is cotranslationally cleaved.…”

“…Two and one N-linked glycans attach to nitrogens of Asn side chains of pro a-and pro a#-subunits and to the b-subunit in the lumen of the ER (Sengupta et al, 1981). These polypeptides are folded in the ER and held together as trimers by noncovalent interactions (Chrispeels et al, 1982;Lelievre et al, 1992). They are then transported via the Golgi and a transport vesicle (dense body) and deposited into the protein storage vacuoles (PSVs).…”

“…They are then transported via the Golgi and a transport vesicle (dense body) and deposited into the protein storage vacuoles (PSVs). The propeptides of pro a-and pro a#-subunits are cleaved after assembly into trimers (Lelievre et al, 1992). Thus far, the intracellular compartment or the processing enzymes, which cleave the propeptide, have not been identified.…”