Chloroplast import characteristics of chimeric proteins

We have examined the import of a series of chimeric precursor proteins into chloroplasts. These fusion proteins contained the transit peptide, and various amounts of the amino-terminal region of the mature peptide, from the small subunit of ribulose 1,5-bisphosphate carboxylase, linked to the coat protein of brome mosaic virus. Chimeric genes were cloned into SP6 plasmids and in vitro transcription/translation was used to produce fusion proteins, which were examined in a quantitative in vitro import assay. A chimeric protein which contained only the transit peptide fused to the coat protein was imported into chloroplasts. A second chimeric precursor, which also contained a small portion of the mature peptide, was imported at nearly the same rate. A chimeric protein which contained the transit peptide and most of the mature peptide fused to the coat protein was not imported. These results suggest that secondary or tertiary structural features of precursor proteins are important for protein import, and that the presence of a transit peptide in a protein does not necessarily ensure import of that protein into chloroplasts.