Poster: Organelle Biogenesis

Abs # 1157: Analysis of Toc complex on chloroplast outer envelope membrane by Blue Native PAGE: A functionally active protein import complex

Presenter:	Kikuchi, Shingo , skikuchi@protein.osaka-u.ac.jp
Authors	Kikuchi, Shingo  (A)   Hirohashi, Toshiya  (A)   Nakai, Masato  (A)
Affiliations:	(A): Inst. for Prot. Res., Osaka Univ.

Most chloroplast proteins are encoded by the nuclear genome and synthesized in the cytosol as a preprotein which contains an amino-terminal transit peptide. Protein translocation across the outer and inner envelope membranes of chloroplast is mediated by the Toc and Tic complexes, respectively. We reported previously that pea chloroplast Toc complexes consisting of at least Toc34, Toc75 and Toc159 were migrated at the sizes ranged from 700 to 800 kDa on Blue Native PAGE. To elucidate molecular architecture of Toc complexes further, we determined the amount of each Toc components in the isolated chloroplasts by comparing prepared standard proteins and estimated their stoichiometry. Restricted proteolysis of the intact chloroplasts caused partial dissociation of some Toc components and revealed core components of the Toc complex. Isolated intact chloroplasts were incubated with radio-labelled precursor proteins and separated by Blue Native PAGE. Signal was detected at the sizes ranged from 700 to 800 kDa. This indicates that 700 to 800 kDa Toc complexes we demonstrated are genuine functionally active protein import complexes. Also, we have obtained several Arabidopsis mutants of Toc genes. We are currently analyzing whether the Toc complex of mutant chloroplasts show any different molecular architecture as compared to that of the wild-type.