Intracellular immunolocalization of adenosine 5′-diphosphoglucose pyrophosphorylase in developing endosperm cells of maize (Zea mays L.)

Abstract

We present immuno-electron microscopic evidence to show that ADPglucose pyrophosphorylase (AGP, EC 2.7.7.27) encoded by the Sh2 (shrunken 2) and the Bt2 (brittle 2) genes is localized to amyloplasts in developing endosperm of maize. The three AGP antibodies, including the two maize antisera, each raised against the Sh2encoded large or the Bt2-encoded small subunit and the spinach leaf protein, showed strong immuno-gold signals on developing starch grains in amyloplasts. The maize antibodies, but not the spinach, detected additional cross-reactivity sites to endosperm cell wall. Similar endosperm sections of the sh2-null mutant, lacking the Sh2-encoded subunit, yielded a drastic reduction in immuno signal on both starch grains and cell wall with the Sh2 anti-serum. However, the Bt2 and the spinach antisera showed no detectable difference between the sh2-null and the wild-type genotypes, except that the spinach antisera showed no reactivity to the cell wall in either of the two genotypes. Because the Bt2 epitope was readily detectable on the sh2-null starch grains, we suggest that the Bt2 subunit of this heteromeric enzyme is able to target itself to the organelle. The amyloplastic localization of the AGP protein in our studies is given additional significance by recent molecular data which indicate that full-length cDNA clones of the Sh2 and Bt2 genes show no cleavable transit-peptide signal sequence in their deduced aminoacid sequences. The observed disparity between the molecular and immunocytochemical data described here is discussed in the context of other proteins engaged in intracellular translocation with and without the known signal sequences.