ISOLATION OF BIP MUTANTS BY ANALYZING THE DEFECTIVE-ENDOSPERM FUNCTION IN MAIZE

A class of mutations affecting the level of zein synthesis and the fin al size and shape of the endosperm is represented by the viable defect ive endosperms. In this paper, twenty-five de-B mutants were considere d with the aim to describe their effect on the level of b-70 proteins which are members of the 70 kD heat-shock family present in maize prot ein bodies. The b-70 proteins are constitutively expressed at a much h igher level in protein bodies from de-B10, de-B37 and de-B70 mutants. Western blot analysis, with the monoclonal (rat) anti-HSP70 gene famil y antibody, produced against Drosophila HSP70 that cross-react with a wide range of heat-shock cognates of various organisms, provided evide nce to support the assignment of maize b-70s as members of the HSP70 f amily. In maize, the b-70 band can be resolved into three species, whe n analyzed by two-dimensional gel electrophoresis: the two isoforms b- 70I and b-70II are associated with protein body fractions and their ar e derepressed in de-B10, de-B37 and de-B70 mutants. A third protein, b -70III, is present in cytosolic fractions and its level is not influen ced by the three mutations studied. Antibodies specific for b-70I and b-70II proteins were raised against a multiple antigene peptide (MAP) corresponding to the N-terminal region. Western blot analysis indicate s that only the b-70I and b-70II proteins, associated with protein bod ies. gave strong positive signals when probed with MAP. Next, we exami ned the effect of stress conditions in the synthesis of b-70 proteins. Experiments were conducted in vitro with dissected endosperms collect ed at 9 days after pollination. In preparations from tunicamycin-treat ed endosperms, significant amounts of b-70 labeled proteins were easil y detectable. The amounts of labeled proteins were not reduced after a subsequent chase period. Instead, most of the 70 kD proteins were sti ll present after the 5-hours chase period. In contrast, the b-70 prote ins were not responsive to heat-shock. The activity of the b-70 protei ns may also be modulated by their reaction with ATP. The evidences pre sented here demonstrated that the b-70 proteins are similar to a previ ously described protein, the immunoglobulin binding protein BiP. We pr opose a specific role for the de-B10, de-B37 and de-B70 mutations in t he ER-metabolism.