A role for gamma 3 hordein in the transport and targeting of prolamin polypeptides to the vacuole of developing barley endosperm.
Hordein synthesis, transport and deposition was analysed by immunocytochemistry in developing endosperm cells of wild-type (Carlsberg II) and mutant varieties deficient in B hordein (hor2ca), gamma 1 hordein (Donetsky), gamma 2 hordein and minor B hordein polypeptides (Haisa), or gamma 3 hordein (Nevsky). In all varieties, hordein polypeptides were detected both in the cytoplasm as globules, ranging in diameter from 50 nm to 1.24 microns, and in the vacuole as protein bodies. In the cytoplasmic globules B and C hordein polypeptides are assembled as a core and are surrounded by an outer layer of gamma 1 and gamma 2 hordein. The globules apparently fuse several times in the cytoplasm before entering the vacuole. Absence of gamma 3 hordein in the mutant Nevsky leads to a dramatic change in hordein polypeptide targeting, the hordein storage proteins being largely deposited in the lumen of the rough endoplasmic reticulum. gamma 3 Hordein is unique among the sulphur-rich hordein polypeptides, being monomeric and forming only intramolecular disulphide bridges, while the other B and gamma hordein polypeptides are aggregated by intermolecular disulphide bridges. Retention of hordein in the rough endoplasmatic reticulum in the absence of gamma 3 hordein suggests that gamma 3 hordein may maintain the prolamin storage polypeptides in a transport competent state. The sequence of the mature gamma 3 hordein polypeptide was deduced from a cDNA clone, and compared with gamma 2 hordein. The epitope recognized by the gamma 1 + gamma 2 hordein-specific BX monoclonal antibody used for immunocytochemistry was mapped to include E190 and K193, by synthesizing overlapping oligopeptides.
Rechinger
K B
KB
Department of Physiology, Carlsberg Laboratory, Valby, Denmark.
Simpson
D J
DJ
Svendsen
I
I
Cameron-Mills
V
V
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001692:Biological Transport; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D000939:Epitopes; D005983:Glutens; D001467:Hordeum / Q000378:metabolism* / Q000648:ultrastructure; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D015388:Organelles / Q000737:chemistry / Q000648:ultrastructure; D010449:Peptide Mapping; D010940:Plant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D055315:Prolamins; D012639:Seeds / Q000254:growth & development* / Q000648:ultrastructure; D017421:Sequence Analysis; D014617:Vacuoles / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D010940:Plant Proteins; D055315:Prolamins; D005983:Glutens