Cell-free synthesis of leaf protein: Identification of an apparent precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase

The selective transport of cytoplasmically synthesized organelle proteins, like animal secretory proteins, may be achieved via the production of precursor polypeptides, as indicated by ion exchange chromatography of their tryptic peptides.

Abstract

Cytoplasmic mRNA has been isolated from the leaves of pea seedlings. Translation of this RNA in the wheat germ cell-free system produces two major products, RI and RII, with molecular weights of 33,000 and 20,000, respectively. Both of these products are considerably larger than the small subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39], which is the major product of cytoplasmic protein synthesis in vivo and has a molecular weight of 14,000. Antiserum prepared against the small subunit of ribulose-1,5-bisphosphate carboxylase precipitates from the cell-free products, in 2-3% yield, three polypeptides of molecular weights 18,000, 16,000 and 14,000. The smallest of these polypeptides is indistinguishable, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, from the small subunit of ribulose-1,5-bisphosphate carboxylase. Although the cell-free product RII is not precipitated with antiserum prepared against the small subunit of ribulose-1,5-bisphosphate carboxylase, the two polypeptides do show extensive sequence homology, as indicated by ion exchange chromatography of their tryptic peptides. The production of RII can also be achieved in a polysome-primed cell-free system, where protein synthesis is restricted to the completion of polypeptide chains that have already been initiated in vivo. These results indicate that RII is apparently a precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase. We suggest that the selective transport of cytoplasmically synthesized organelle proteins, like animal secretory proteins, may be achieved via the production of precursor polypeptides.