Accumulation of beta-Conglycinin in Soybean Cotyledon through the Formation of Disulfide Bonds between alpha %26apos;- and alpha-Subunits

作者：Wadahama Hiroyuki; Iwasaki Kensuke; Matsusaki Motonori; Nishizawa Keito; Ishimoto Masao; Arisaka Fumio; Takagi Kyoko; Urade Reiko^*

来源：Plant Physiology, 2012, 158(3): 1395-1405.

DOI：10.1104/pp.111.189621

beta-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean beta-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the alpha%26apos;- and alpha-subunits of beta-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the beta-conglycinin complexes containing the disulfide-linked alpha%26apos;/alpha-subunits were complexes of more than 720 kD. The alpha%26apos;- and alpha-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between alpha%26apos;/alpha-subunits residing in different beta-conglycinin hexamers, but the binding of P34 to alpha%26apos;- and alpha-subunits reduces the linkage between beta-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when beta-conglycinin was expressed under nonreducing conditions.

• 出版日期2012-3