A ROLE FOR GAMMA-3 HORDEIN IN THE TRANSPORT AND TARGETING OF PROLAMINPOLYPEPTIDES TO THE VACUOLE OF DEVELOPING BARLEY ENDOSPERM

Hordein synthesis, transport and deposition was analysed by immunocyto chemistry in developing endosperm cells of wild-type (Carlsberg II) an d mutant varieties deficient in B hordein (hor2ca), gamma1 hordein (Do netsky), gamma2 hordein and minor B hordein polypeptides (Haisa), or g amma3 hordein (Nevsky). In all varieties, hordein polypeptides were de tected both in the cytoplasm as globules, ranging in diameter from 50 nm to 1.24 mum, and in the vacuole as protein bodies. In the cytoplasm ic globules B and C hordein polypeptides are assembled as a core and a re surrounded by an outer layer of gamma1 and gamma2 hordein. The glob ules apparently fuse several times in the cytoplasm before entering th e vacuole. Absence of gamma3 hordein in the mutant Nevsky leads to a d ramatic change in hordein polypeptide targeting, the hordein storage p roteins being largely deposited in the lumen of the rough endoplasmic reticulum. Gamma3 Hordein is unique among the sulphur-rich hordein pol ypeptides, being monomeric and forming only intramolecular disulphide bridges, while the other B and gamma hordein polypeptides are aggregat ed by intermolecular disulphide bridges. Retention of hordein in the r ough endoplasmatic reticulum in the absence of gamma3 hordein suggests that gamma3 hordein may maintain the prolamin storage polypeptides in a transport competent state. The sequence of the mature gamma3 hordei n polypeptide was deduced from a cDNA clone, and compared with gamma2 hordein. The epitope recognized by the gamma1 + gamma2 hordein-specifi c BX monoclonal antibody used for immunocytochemistry was mapped to in clude E190 and K193, by synthesizing overlapping oligopeptides.