Accumulation of β-conglycinin in soybean cotyledon through the formation of disulfide bonds between α'- and α-subunits.
β-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 β-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 α'- and α-subunits of β-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 β-conglycinin complexes containing the disulfide-linked α'/α-subunits were complexes of more than 720 kD. The α'- and α-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 α'/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α'- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions.
Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Hiroyuki
H
Wadahama
Kensuke
K
Iwasaki
Motonori
M
Matsusaki
Keito
K
Nishizawa
Masao
M
Ishimoto
Fumio
F
Arisaka
Kyoko
K
Takagi
Reiko
R
Urade
D052179:Antigens, Plant / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002850:Chromatography, Gel; D018548:Cotyledon / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005916:Globulins / Q000302:isolation & purification / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011485:Protein Binding; D055314:Seed Storage Proteins / Q000302:isolation & purification / Q000378:metabolism*; D030262:Soybean Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013025:Glycine max / Q000378:metabolism*
D052179:Antigens, Plant; D004220:Disulfides; D005916:Globulins; D046912:Multiprotein Complexes; D055314:Seed Storage Proteins; D030262:Soybean Proteins; C013894:beta-conglycinin protein, Glycine max; C011474:glycinin