The role of the endoplasmic reticulum in the synthesis and transport of ?-amylase in barley aleurone layers

Abstract

The subcellular site of α-amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA3). Using [(35)S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA3 was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker α-amylase. This protein was identified as authentic α-amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized α-amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled α-amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of α-amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA3 were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA3-treated aleurone layers, five peaks of α-amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA3 only three peaks of activity were found. In incubation media, four isoenzymes were found after GA3 treatment and two were found after incubation without GA3. We conclude that at least five α-amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.