A mammalian cytochrome fused to a chloroplast transit peptide is a functional haemoprotein and is imported into isolated chloroplasts

engineered by further N-terminal linkage of a prokaryotic secretory signal. Expression of this tripartite fusion resulted in mg quantities of the sign al sequence-processed Tp-Cyt protein, which was eventually targeted to the membranes. The chromogenic nature of the chirnaera and its localization to the bacterial membrane facilitated the biochemical isolation of the precurs or in a soluble and functional form. The purified preprotein displayed spec tral and enzymic properties that were indistinguishable from the native par ental Cyt, implying an absence of observable influence of the Tp on the con formation of the haemoprotein. The chimaeric precursor was imported into th e stroma of the isolated chloroplasts in a dose-dependent manner. Import wa s also strongly dependent upon exogenously supplied ATP. The stromally impo rted chimaeric precursor protein was processed to a size characteristic of Cyt, The small subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) is a major chloroplast stromal protein that is cytosolically synt hesized as a precursor with an N-terminal extension, known as the transit s equence or transit peptide (Tp), The Tp is essential for the post-translati onal uptake of the precursor by the chloroplast. The Tp is thought to influ ence the conformation of the precursor protein and to facilitate polypeptid e translocation across the chloroplast envelope barrier via a Tp-selective translocon, To address these issues we have devised a novel strategy to gen erate substrate amounts of a chloroplast targeting sequence as a fusion wit h the chromogenic globular domain of cytochrome b(5) (Cyt). The chimaeric p rotein is an ideal probe for investigating the conformation of a preprotein and events surrounding protein import into isolated chloroplasts. The Cyt of liver endoplasmic reticulum was fused at its N-terminus with the Tp of t he small subunit of Rubisco of Pisum sativum (pea). To enhance its producti on by clearance from the cytoplasm of Escherichia coli, the chimaera was en gineered by further N-terminal linkage of a prokaryotic secretory signal. E xpression of this tripartite fusion resulted in mg quantities of the signal sequence-processed Tp-Cyt protein, which was eventually targeted to the me mbranes. The chromogenic nature of the chimaera and its localization to the bacterial membrane facilitated the biochemical isolation of the precursor in a soluble and functional form. The purified preprotein displayed spectra l and enzymic properties that were indistinguishable from the native parent al Cyt, implying an absence of observable influence of the Tp on the confor mation of the haemoprotein. The chimaeric precursor was imported into the s troma of the isolated chloroplasts in a dose-dependent manner. Import was a lso strongly dependent upon exogenously supplied ATP. The stromally importe d chimaeric precursor protein was processed to a size characteristic of Cyt .