Poster: Organelle Biogenesis

Abs # 1151: Application of Mutant Precursors for the Analysis of Interaction between Precursors and Protein Translocation Machinery during Protein Import into Chloroplasts

Presenter:	Akita, Mitsuru , akita@agr.ehime-u.ac.jp
Authors	Akita, Mitsuru  (A)
Affiliations:	(A): Faculty of Agriculture, Ehime University

Most chloroplastsic proteins are encoded in the nuclear genome and synthesized as precursors with targeting sequences at their N-termini. These proteins are then transported into chloroplasts utilizing proteinacious translocation machinery embedded in both the outer and the inner envelope membranes. Though many components involved in protein import have been identified, there yet to be unclear whether other components are necessary. On the other hand, other important issues, such as, how precursors sequentially interact with components or whether complex associate and dessociate during protein translocation, are not well understood. To address these problems, I have been trying to obtain the various translocation intermediates, with precursors trapped in the machinery at the various steps during protein translocation by the application of chemically modified precusors. In detail, a set of mutant precursors that has single cystein residues at the various position were designed and obtained as recombinant proteins. These mutants were then modified with SH-reactive reagents, which might plug the protein channel due to their steric hindrance. Import of biotinylated precursors were not affected by the modification, but were inhibited when avidin was incubated with precursors prior to the import reaction. This indicates that the translocation intermediates were formed. Currently the analysis of these traslocation intermediates are underway.