ALPHA-AMYLASE ISOFORMS ARE POSTTRANSLATIONALLY MODIFIED IN THE ENDOMEMBRANE SYSTEM OF THE BARLEY ALEURONE LAYER

The subcellular site of the posttranslational modification of alpha-am ylase was investigated in aleurone layers of barley the (Hordeum vulga re L. cv Himalaya). Aleurone layers of Himalaya barley synthesize and secrete two groups of alpha-amylase isoforms, referred to as low-isoel ectric point (low-pl) or HAMY1 and high-pl or HAMY2, when incubated in gibberellic acid and CaCl2. Whereas homogenates of aleurone layers co ntain four isoforms of HAMY1 with pls 4.90, 4.72, 4.64, and 4.56, incu bation media contain predominantly isoforms 4.72 and 4.56. Microsomal membranes isolated from aleurone layers contain all four isoforms of H AMY1. Microsomal membranes can be resolved into two peaks by isopycnic density gradient centrifugation: a peak of heavy membranes with endop lasmic reticulum and Golgi apparatus (GApp) marker enzyme activities a nd a peak of light membranes with characteristics of the GApp. The hea vy membranes contain proportionally more HAMY1 pl 4.90 and 4.64 isofor ms, whereas light membranes contain a higher proportion of pl 4.72 and 4.56 isoforms. Experiments with the ionophore monensin show that memb ranes of the GApp as well as the endoplasmic reticulum are involved in the posttranslational modification of HAMY1 isoforms. Monensin inhibi ts the secretion of alpha-amylase and causes the enzyme to accumulate within the cell. Precursor forms of HAMY1 accumulate in light membrane s isolated from monensin-treated aleurone layers indicating that the G App is involved in the conversion of the precursor of the secreted for ms of the enzyme.