SURFACE LOCALIZATION OF ZEIN STORAGE PROTEINS IN STARCH GRANULES FROMMAIZE ENDOSPERM - PROTEOLYTIC REMOVAL BY THERMOLYSIN AND IN-VITRO CROSS-LINKING OF GRANULE-ASSOCIATED POLYPEPTIDES

Starch granules from maize (Zea mays) contain a characteristic group o f polypeptides that are tightly associated with the starch matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight, G.W. Sing letary, 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 d etermined. Proteolysis of starch granules at subgelatinization tempera tures using the thermophilic protease thermolysin led to selective rem oval of the zeins, whereas granule-associated proteins of 32 kD or abo ve, including the waxy protein, starch synthase I, and starch-branchin g enzyme IIb, remained refractory to proteolysis. Granule-associated p roteins from maize are therefore composed of two distinct classes, the surface-localized zeins of 10 to 27 kD and the granule-intrinsic prot eins of 32 kD or higher. The origin of surface-localized delta-zein wa s probed by comparing delta-zein levels of starch granules obtained fr om 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 in dicating that delta-zein adheres to granule surfaces after disruption of the amyloplast envelope. Cross-linking experiments show that the ze ins are deposited on the granule surface as aggregates. In contrast, t he granule-intrinsic proteins are prone to covalent modification, but do not form intermolecular cross-links. We conclude that individual gr anule intrinsic proteins exist as monomers and are not deposited in th e form of multimeric clusters within the starch matrix.