FUNCTIONAL DOMAINS OF THE FERREDOXIN TRANSIT SEQUENCE INVOLVED IN CHLOROPLAST IMPORT

In order to analyze the information content of a chloroplast transit sequence, we have constructed and analyzed by in vitro assays seven substitution and 20 deletion mutants of the ferredoxin transit sequence. The N-terminal part and the C-terminal part are important for targeting, and in addition the C-terminal region is required for processing. A third region is important for translocation but not for the initial interaction with the envelope. A fourth region is less essential for in vitro import. Purified precursors were tested for their ability to compete for the in vitro import of radiolabeled wildtype precursor, which confirmed the important role in chloroplast recognition of both the N- and the C-terminal domain of the transit sequence. Monolayer experiments showed that the N terminus was mainly involved in the insertion into mono-galactolipid-containing lipid surfaces whereas the C terminus mediates the recognition of negatively charged lipids. A sequence comparison to other transit sequences suggests that the domain structure of the ferredoxin transit sequence can be extended to these sequences and thus reveals a general structural design of transit sequences.