BT1, A STRUCTURAL GENE FOR THE MAJOR 39-44 KDA AMYLOPLAST MEMBRANE POLYPEPTIDES

The aim of the present work was to investigate the relationship betwee n the Bt1 gene (Bt1) and the major 39-44 kDa amyloplast membrane polyp eptides which were deficient in amyloplast membranes of brittlel (bt1) kernels of maize (Zea mays L,). A rapid yet gentle procedure for the isolation of amyloplasts from immature kernels is described. These amy loplasts were relatively free of contamination by other cellular compo nents, and immunological studies showed that they contained polypeptid es which reacted with antibodies to maize starch blanching enzyme and ADP-Glc pyrophosphorylase. Purified membranes isolated from the amylop last contained a polypeptide which reacted with antibodies to the P-tr anslocator from spinach chloroplasts. However, a cluster of 39-44 kDa polypeptides accounted for about 40% of the total amyloplast membrane protein from W64A kernels, These polypeptides were specifically recogn ized by antibodies raised against a fusion protein consisting of 56 am ino acids of the carboxyl terminus of the BT1 protein and glutathione S-transferase, The BT1 antibodies also reacted with the abundant polyp eptides in amyloplast membranes from hybrid kernels (Doebler 66XP and Pioneer 3780), and the shrunken1 and shrunken2 mutant genotypes, but n o BT1 reacting polypeptides were present in amyloplast membranes from bt1 mutant kernels, We were unable to detect BT1 by the immunoblot pro cedure in microsomal membranes from embryo and pericarp tissues from t he kernel, from seedling roots and shoots, or in membranes from mitoch ondria and chloroplasts. The same BT1 immunoblot pattern was obtained for proteins extracted from microsomal membranes from developing endos perm and from purified amyloplast membranes. A linear relationship bet ween the number of copies of Bt1 alleles and the levels of BT1 in endo sperm microsomal membranes was demonstrated in a gene dosage series. B T1 was not extracted from amyloplast membranes by chloroform/methanol or by alkaline buffer at pH 11.5, but was partially extracted by 0.1 M NaOH. These lines of evidence support the conclusion that Bt1 is the structural gene for the major 39-44 kDa amyloplast membrane polypeptid es and that these polypeptides are integral proteins specific to amylo plast membranes from the endosperm.