Sequestration of Pea Reserve Proteins by Rough Microsomes

Abstract

Free polysomes, polysomes released from membranes and rough microsomal vesicles isolated from developing cotyledons of P. sativum L. cv. Burpeeana were used to direct cell-free protein synthesis in a wheat germ system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] demonstrated that the polypeptide products had MW ranging from 12,000-74,000. Some of the polypeptides migrated during electrophoresis with the same mobility as polypeptides present in legumin and vicilin preparations. By the use of rabbit antibodies raised against pea reserve proteins it was estabilshed that polysomes released from membranes and rough microsomes directed the synthesis of polypeptides that were related to reserve proteins whereas free polysomes did not. Centrifugation studies indicated that the majority of the radioactivity incorporated by rough microsomes was specifically associated with the microsomes. The incorporated radioactivity of sedimented microsomes was not released by treatment with KCl and was resistant to proteolysis unless detergent was present. SDS-PAGE revealed that the sequestered translation products were related to pea reserve proteins. Evidently, the reserve proteins of pea cotyledons are synthesized exclusively by membrane-bound polysomes and they are sequestered within the rough endoplasmic reticulum.