Precursor Forms of Pea Vicilin Subunits: MODIFICATION BY MICROSOMAL MEMBRANES DURING CELL-FREE TRANSLATION

Abstract

Polyribosomal RNA isolated from pea cotyledons at various developmental stages programmed the cell-free synthesis of polypeptides which were recognized by antibodies specific for pea storage proteins. There were quantitative and qualitative changes in the template activity during seed maturation. Most of the polysomal RNA was associated with the membrane fraction, and all of the template for storage protein occurred in this fraction. Using RNA from a stage of seed maturation at which the synthesis of the high-molecular weight vicilin polypeptides predominate, it was found that the major translation products, although antigenically recognizable as storage protein, did not coincide with the authentic vicillin polypeptides on denaturing polyacrylamide gels. The addition during translation of microsomal membranes from dog pancreas or pea cotyledons resulted in the appearance of new polypeptides which did coincide with some of the authentic vicilin polypeptides (in the apparent molecular weight regions of 75,000 and 50,000) and were antigenically recognizable as storage protein. Other translation products related to storage protein were not visibly altered in their electrophoretic mobility by the addition of membranes. Microsomal membranes treated with Triton X-100 were not effective in modifying the cell-free products. The modified vicilin polypeptides and at least two other translation products were protected from proteolytic degradation, suggesting that they were sequestered within microsomal vesicles. Thus, these storage protein components may be synthesized by a mechanism analogous to that described for membrane and secretory proteins (Blobel G, B Dobberstein 1975 J Cell Biol 67: 835-851).