Surface Localization of Zein Storage Proteins in Starch Granules from Maize Endosperm1

Abstract

Starch granules from maize (Zea mays) contain a characteristic group of polypeptides that are tightly associated with the starch matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight, G.W. Singletary, P.L. Keeling, B.P. Wasserman [1996] Plant Physiol 111: 821-829). Zeins comprise about 50% of the granule-associated proteins, and in this study their spatial distribution within the starch granule was determined. Proteolysis of starch granules at subgelatinization temperatures using the thermophilic protease thermolysin led to selective removal of the zeins, whereas granule-associated proteins of 32 kD or above, including the waxy protein, starch synthase I, and starch-branching enzyme IIb, remained refractory to proteolysis. Granule-associated proteins from maize are therefore composed of two distinct classes, the surface-localized zeins of 10 to 27 kD and the granule-intrinsic proteins of 32 kD or higher. The origin of surface-localized delta-zein was probed by comparing delta-zein levels of starch granules obtained from homogenized whole endosperm with granules isolated from amyloplasts. Starch granules from amyloplasts contained markedly lower levels of delta-zein relative to granules prepared from whole endosperm, thus indicating that delta-zein adheres to granule surfaces after disruption of the amyloplast envelope. Cross-linking experiments show that the zeins are deposited on the granule surface as aggregates. In contrast, the granule-intrinsic proteins are prone to covalent modification, but do not form intermolecular cross-links. We conclude that individual granule intrinsic proteins exist as monomers and are not deposited in the form of multimeric clusters within the starch matrix.

Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry

“…Marker enzyme assays confirmed that our whole amyloplast organelle preparations contain very little extraneous material from the surrounding tissue. All of the proteins we identified were typical of amyloplast tissue [30][31][32], and no obvious contaminants from other organelles were identified. Membrane fractions isolated from this preparation yielded a small subset of proteins that overlap with the intact amyloplast preparations, as well as a large percentage of functionally distinct proteins that are characteristic of specialized membrane functions such as transport and receptor signaling.…”

Localization of a bacterial protein in starch granules of transgenic maize kernels

“…The approach was based on the fact that polypeptides within the starch granules are not susceptible to hydrolysis on treatment of intact granules with exogenous proteases (16). Starch samples were isolated from individual mature kernels of the transgenic maize line carrying construct P77, using three different levels of preparation stringency (75% ethanol, 95% ethanol, or 75% ethanol plus 3% ␤-mercaptoethanol) to remove external proteins.…”

“…Although the import of nucleus-encoded proteins into chloroplasts has been studied extensively (25), the mechanism of import of nucleus-encoded proteins into amyloplasts has not been established (16). Chloroplast-targeted proteins are synthesized with transit peptides usually removed upon import into the chloroplast.…”

“…To remove exogenous proteins from purified starch or ground maize kernel, samples were incubated with 5 mM CaCl 2 solution containing 100 g/ml thermolysin (Sigma) at 64°C for Ϸ16 h (16). The reactions were terminated by the addition of EDTA to 20 mM.…”

Regulation of starch accumulation by granule-associated plant 14-3-3 proteins

“…Reduction in GF14 and protein levels in the starch granules of antisense plants and presence of 14-3-3 proteins in commercial corn starch. Isolated starch granules from wild-type and antisense Arabidopsis were treated with thermolysin to remove externally attached proteins and subjected to SDS͞PAGE Western analysis with 14-3-3 protein antibodies (29). Protein extracts from 3 mg of starch from wild-type (lanes 1 and 2), GF14 antisense (lane 3), and GF14 antisense (lane 4) plants were probed with antibodies recognizing GF14 (lanes 1 and 3) and (lanes 2 and 4).…”