In vitro uptake and processing of prezein and other maize preproteins by maize membranes.

Abstract

A cell-free, mRNA-dependent system was developed for the translation and processing of zein preproteins. A rough endoplasmic reticulum (RER)-enriched fraction, isolated by sucrose density gradients, can be treated with micrococcal nuclease to destroy endogenous messages. When these membranes are added to a wheat germ protein-synthesizing system together with zein mRNA, synthesis and processing of the polypeptides to the mature products takes place. The RER fraction from the endosperm has a different protein composition than that prepared from either the shoot or nucellar tissue and processes prezein more efficiently. The cleavage of the preproteins appears to be a cotranslational step as the completed preprotein chains cannot be processed, although they can be taken up to a limited extent. This small uptake, or absorption, of unprocessed zein seems to be an artifact and may be related to the unusual solubility properties of zein. A sodium dodecyl sulfate (SDS)-urea polyacrylamide gel system was developed which is particularly suited for the separation of low MW proteins (< 10,000 daltons). Using this method, the products of in vitro zein processing were examined and no presequence polypeptides were detected. Evidently, the zein cleavage proteinase is probably an exopeptidase.