PARENT SESSION

Symposium S7C Biosynthesis and assembly: Protein trafficking
Thursday September 2nd, 2004 2:40 PM-4:40 PM Room 510B
Chair: Ken Cline
Co-Chair: Steven Theg

Characterization of the Preprotein Translocon at the Outer Membrane of Chloroplasts by Blue Native PAGE. Shingo Kikuchi^*,1, Toshiya Hirohashi¹, Masato Nakai¹, ¹ Inst. Protein Res., Osaka Univ., Japan

ABSTRACT- Most chloroplastic proteins are encoded by nuclear genes and synthesized in the cytosol as a precursor protein which contains an amino-terminal transit peptide. These proteins are then post-translationally imported across the double membrane of chloroplasts. The chloroplastic outer and inner envelope membranes contain multisubunit machineries for the import of precursor proteins, termed Toc and Tic complexes, respectively. For the constituents of the Toc complex in pea chloroplasts, four proteins (Toc75, Toc159, Toc34, and Toc64) have been reported to be involved in this process. Although considerable information has been accumulated about the functions of individual Toc components, the molecular architecture and organization of the Toc complex itself remain to be elucidated.
In this study, we have analyzed the molecular architecture and organization of the Toc complex by Blue Native PAGE (BN-PAGE), which is a high-resolution method for separating membrane protein complexes under non-denaturing conditions. Pea chloroplasts were directly solubilized in detergent solution and analyzed by BN-PAGE. Immunoblot analyses showed that Toc75, Toc159, and Toc34 forms a highly stable complex whose molecular mass is approximately 800 kDa. Selective proteolysis revealed a core complex of the Toc complex which is resistant to proteases and detergent treatments. Furthermore, a combination of in vitro precursor protein binding assays and BN-PAGE showed a chaseable import intermediate complex.

KEY WORDS: protein transport, chloroplast, BN-PAGE