Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides i n the protein bodies of developing pea cotyledons

Abstract

Cotyledons of developing pea seeds (Pisurn sativum L.) were labeled with radioactive amino acids and glucosamine, and extracts were prepared and separated into fractions rich in endoplasmic reticulum (ER) or protein bodies. The time-course of synthesis of the polypeptides of legumin and vicilin and the site of their assembly into protein oligomers were studied using immunoaffinity gels and sucrose density gradients. When cotyledons were pulselabeled (1-2 h), newly synthesized legumin was present in polypeptides with Mr 60,000-65,000, and newly synthesized vicilin was present as a series of polypeptides with Mr 75,000, 70,000, 50,000, and 49,000. These radioactive polypeptides were found primarily in the ER (Chrispeels et al., 1982, J. Cell BioL, 93: 5-14). During a subsequent chase period, newly synthesized reserve proteins were initially present in the protein bodies in the above-named polypeptides. Between 1 and 20 h later, radioactive legumin subunits (Mr 40,000 and 19,000) and smaller vicilin polypeptides (Mr 34,000, 30,000, 25,000, 18,000, 14,000, 13,000, and 12,000) appeared in the protein bodies. The appearance of these labeled polypeptides in the protein bodies was not the result of a slow transport from the ER (or cytoplasm). Newly synthesized legumin and vicilin polypeptides were assembled into oligomers of 85 and 7S, respectively, in the ER. They appeared in the protein bodies in these oligomeric forms before the appearance of the smaller polypeptides (Mr <49,000). These results indicate that the smaller vicilin polypeptides (Mr <49,000) arise by delayed posttranslational processing of some or all of the larger vicilin polypeptides. The precursors of legumin are completely processed in the protein bodies 2-3 h after their synthesis. The processing of the vicilin precursors is much slower (6-20 h) and only a fraction of the precursor molecules are processed. As a result both large (Mr >49,000) and small polypeptides of vicilin accumulate in the protein bodies, whereas legumin accumulates only as polypeptides of Mr 40,000 and 19,000. © 1982, Rockefeller University Press., All rights reserved.