Co-ordinated expression of multiple enzymes in different subcellular compartments in plants

A gene expression system designed for coordinated expression of multiple genes in plants and their targeting to specified subcellular locations was tested. A series of genes encoding polyproteins containing the tobacco vein mottling virus (TVMV) NIa proteinase along with two other reporter genes (those encoding the Escherichia coli acetate kinase (ACK) and Tn9 chloramphenicol acetyl transferase (CAT) enzymes) were assembled. The respective coding sequences of these genes were separated by a TVMV NIa proteinase recognition sequence. In addition, in some instances, chloroplast targeting information [a transit peptide (TP) from a pea rbcS gene) was incorporated into the polyprotein. We found that the NIa proteinase can be used to express, as individual polypeptides, the ACK and CAT proteins, and that these proteins retain enzymatic activity. Polyproteins with the structure TP-NIa-ACK-CAT or TP-ACK-CAT-NIa failed to yield chloroplast-localized ACK and CAT proteins, although the latter did give rise to a chloroplast-localized ACK-CAT polyprotein. These results indicate that the NIa proteinase acts in cis more rapidly than transport of proteins into the chloroplast, but that chloroplast localization can take place before complete processing of the polyprotein. Polyproteins with the structures ACK-NIa-TP-CAT and TP-ACK-NIa-TP-CAT yielded appropriately processed and targeted ACK and CAT. Our results show that subcellular localization signals can be effectively recognized in the context of a polyprotein, and they suggest an appropriate strategy far simultaneous engineering of multiple subcellular compartments in plants.