Surface Localization of Zein Storage Proteins in Starch Granules
from Maize Endosperm
: Proteolytic Removal by Thermolysin and in Vitro Cross-Linking of
Granule-Associated Polypeptides |
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Authors: | Chen Mu-Forster and Bruce P Wasserman |
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Institution: | Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick, New Jersey 08901–8520 |
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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 δ-zein was probed by comparing
δ-zein levels of starch granules obtained from homogenized whole
endosperm with granules isolated from amyloplasts. Starch granules from
amyloplasts contained markedly lower levels of δ-zein relative to
granules prepared from whole endosperm, thus indicating that δ-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.It has long been known that starch granules contain bound
polypeptides, with protein levels of isolated starch granules from
maize (Zea mays) ranging from 0.3 to 1.0% based upon
measurement of N2 (May, 1987). A recent study by our
laboratory demonstrates that isolated starch granules from maize
contain several dozen strongly bound polypeptides (Mu-Forster et al.,
1996). The granule-associated proteins include starch-biosynthetic
enzymes such as the waxy protein, SSI, and SBEIIb. These polypeptides
are not removed from intact starch granules by protease treatment or
detergent washing; therefore, they are believed to bind to the starch
and to become irreversibly entrapped within the starch
matrix.Based upon staining intensities of polypeptides extracted from the
starch granule (Mu-Forster et al., 1996), approximately one-half of the
granule-associated proteins in maize consist of low-molecular-mass
polypeptides ranging between 10 and 27 kD. These bands fall within the
size range displayed by the zein storage proteins, however, the spatial
distribution of these polypeptides within the starch granule is
unknown. Zeins have been defined as alcohol-soluble proteins that occur
principally in protein bodies of maize endosperm and that may or may
not require reduction before extraction (Wilson, 1991). The association
of zeins with starch granules during endosperm development would not be
expected because zein genes do not contain transit peptides that would
target these proteins through the amyloplast envelope into the
amyloplast stroma.The objective of this study was to establish the topology of
granule-associated zeins in starch granules from maize endosperm. To
accomplish this, it was necessary to distinguish between
surface-localized and internalized polypeptides. Our working hypothesis
defines polypeptides localized at the starch granule surface as those
that are susceptible to hydrolysis upon treatment of intact granules
with exogenous proteases. Conversely, internal granule proteins are
defined as those that (a) become susceptible to proteolysis only
following thermal disruption of the starch matrix, and (b) resist
extraction by 2% SDS at room temperatures (Denyer et al., 1993; Rahman
et al., 1995; Mu-Forster et al., 1996).In this study we were able to distinguish between surface-localized and
internalized granule-associated polypeptides in starch granules
from maize endosperm by use of the thermophilic protease thermolysin.
Thermolysin is well suited for this purpose because it is highly active
at starch-gelatinization temperatures, and has also been shown to
effectively hydrolyze hydrophobic proteins located at the surfaces of
chloroplasts and other subcellular organelles (Cline et al., 1984; Xu
and Chitnis, 1995). Upon extended incubation of intact starch granules
with thermolysin at subgelatinization temperatures, we found that zeins
were selectively removed from the starch granule surface. All other
granule-associated polypeptides remained inaccessible to proteolytic
attack or to extraction by 2% SDS, unless the starch matrix was first
disrupted by gelatinization. Our results distinguish between the
surface-localized and granule-intrinsic proteins of maize endosperm,
and establish that zeins are localized at the starch-granule surface.
In addition, cross-linking experiments were conducted to determine
nearest-neighbor relationships among zein subunits localized at the
granule surface and granule intrinsic polypeptides localized within the
starch matrix. |
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