Abstract
Following sequestration into the endoplasmic reticulum, wheat high molecular weight glutenin subunits (HMW-GS) assemble into polymers through intermolecular disulfide bond formation. These polymers, which also include low molecular weight glutenin subunits (LMW-GS), have a broad distribution of molecular mass reaching up to several million daltons. To study the mechanism of assembly of the HMW-GS, we have expressed x- and y-type HMW-GS in transgenic tobacco plants. Both types, when expressed individually or in combination, were incorporated into polymers. Partial reduction of polymers formed by different subunits resulted in different patterns of release of homodimers, heterodimers, and monomers. This suggested different arrangements of intermolecular disulfide bonds or different peptide conformations in the vicinity of the disulfide bonds linking x-x, y-y, and x-y type HMW-GS. A mutant of the x-type subunit, lacking a conserved cysteine in the C-terminal domain, assembled into oligomers linked by intermolecular disulfide bonds, but not into large polymers. This mutant was deposited, however, in dense protein bodies, similar to those formed by the native HMW-GS, suggesting that polymer formation and packaging into protein bodies may be the result of different types of interactions. Pulse-chase labeling of proteins in wheat endosperm showed that the assembly of the HMW-GS into insoluble polymers occurs by a slow process which apparently continues after the initiation of protein body formation.
Highlights
(HMW-GS) assemble into polymersthroughintermo- mers linked by intermolecular disulfide bonds, having a molecular disulfide bond formation
The respectant of the x-type subunit, lacking a conservecdysteine tive genes are thought to have diverged from a common proin the C-terminaldomain,assembled into oligomers linked by intermolecular disulfide bonds, but not into large polymers
Time Course of HMW-GS Assembly in Wheat Endosperm-Previous studies from our laboratory had shown that packaga b c de ing of wheat storage proteins into PbBegan shortly after their FIG8. .Partial reduction of polymers formedby the Dx2- subsynthesis and that dense protein bodies (PB) were already evident 4 h after synthesis [31].As our results suggested that HMW-GS may assemble first into oligomers and into largpeolymers, we tested the time course of assembly of the natural HMW-GS from wheat endosperm
Summary
Vol 269, No 12, Issue of March 25, pp. 8924-8930, 1994 Printed in U.S.A. Mechanisms of Assemblyof Wheat High Molecular Weight Glutenins Inferred from Expression of Wild-type and Mutant Subunitisn Transgenic Tobacco*. The respectant of the x-type subunit, lacking a conservecdysteine tive genes are thought to have diverged from a common proin the C-terminaldomain,assembled into oligomers linked by intermolecular disulfide bonds, but not into large polymers. This mutant was deposited, howevienr, dense protein bodies, similatro those formedby the native HMW-GS, suggesting that polymer formation and packaging into protein bodies may be the result of different types of interactions. Following extraction for 2h at room temperature, the extract was centrifuged for 15 min at 27,000 x g and the supernatant was collected and termed the "SDS-soluble HMW-GS." The pellet was interactions betweenx- and y-type subunits or from their pref- re-extracted in the same SDS containing buffer which1%included erentialliberation from the glutenin polymer has notbeen p-ME. Fragment of Dx2 was cloned into a Bluescript K S plasmid (Stratagene, La Jolla,CA) so that the3' end of the HMW-GS fragment was adjacent
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