The import of nuclear-encoded precursor proteins into chloroplasts occurs via a cleavable, N-terminal targeting sequence known as the transit peptide. To test the influence of the mature domain of the small subunit of Rubisco during import in vitro, the precursor (prSSU), the mature domain (mSSU), the transit peptide (SStp) and three Cterminal deletion mutants (Δ52, Δ67, and Δ74) of prSSU were expressed and purified from Escherichia coli. Activity was then evaluated by inhibition of import of 35S-prSSU to show that removal of C-terminal prSSU sequences inhibits its interaction with the translocation apparatus. Import studies demonstrated that prSSU and Δ52 were processed and accumulated within the chloroplast, whereas Δ67 and Δ74 were rapidly degraded. Import-competent proteins were also able to induce anion channel closure for PIRAC (Protein Import Related Iconic C,/u\u3ehannel). Although the C-terminal deletion mutants were less effective at inducing channel closure upon import, they did not affect the mean duration of channel closure. In addition the same proteins, as well as the precursors to the 33 and 23 kD subunits of the oxygen evolving complex of photosystem II, prOE33 and prOE23, respectively, were used in liposome dye release assays to investigate the interaction between precursors and chloroplast outer membrane lipids. Chloroplast precursor proteins do interact with liposomes mimicking the chloroplast outer envelope lipids through a process mediated through the transit peptide and requiring the presence of non-bilayer forming lipids and anionic lipids. The interaction of the transit peptide with liposomes involves electrostatic interactions between the peptide and the anionic lipids in the liposome. From this study, two additional precursors were shown to be membrane active, prOE33 and prOE23 Finally to investigate the in vivo activity of prSSU transit peptide, we designed green fluorescent protein (GPP) chimeras with the precursor to the small subunit of Rubisco (prSSU-GFP). GFP alone or prSSU-GFP could be expressed in a transient assay in onion epidermal cells. These experiments lay the groundwork for expression of mutant precursors fused to GFP in either onion epidermal to explore the effect mutations in the transit peptide have on targeting and import relative to the wildtype precursor
