Btl, a structural gene for the major 39–44 kDa amyloplast membrane polypeptides

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Abstract
The aim of the present work was to investigate the relationship between theBtlgene(Btl)and the major 39–44 kDa amyloplast membrane polypeptides which were deficient in amyloplast membranes ofbrittlel (btl)kernels of maize (Zea maysL.). A rapid yet gentle procedure for the isolation of amyloplasts from immature kernels is described. These amyloplasts were relatively free of contamination by other cellular components, and immunological studies showed that they contained polypeptides which reacted with antibodies to maize starch branching enzyme and ADP‐Gle pyrophosphorylase. Purified membranes isolated from the amyloplast contained a poly‐peptide which reacted with antibodies to the Pi‐translocator 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 recognized by antibodies raised against a fusion protein consisting of 56 amino acids of the carboxyl terminus of the BTI protein and glutathione S‐transferase. The BT1 antibodies also reacted with the abundant polypeptides in amyloplast membranes from hybrid kernels (Doebler 66XP and Pioneer 3780), and theshrunkenlandshrunken2mutant genotypes, but no BTl reacting polypeptides were present in amyloplast membranes frombtlmutant kernels. We were unable to detect BTl by the immunoblot procedure in microsomal membranes from embryo and pericarp tissues from the kernel, from seedling roots and shoots, or in membranes from mitochondria and chloroplasts. The same BTl immunoblot pattern was obtained for proteins extracted from microsomal membranes from developing endosperm and from purified amyloplast membranes. A linear relationship between the number of copies ofBtlalleles and the levels of BTl in endosperm microsomal membranes was demonstrated in a gene dosage series. BTl was not extracted from amyloplast membranes by chloroform/methanol or by alkaline buffer at pH 11.5, but was partially extracted by 0.1MNaOH. These lines of evidence support the conclusion thatBtl is the structural genefor the major 39–44 kDa amyloplast membrane polypeptides and that these polypeptides are integral proteins specific to amyloplast membranes from the endosperm.