Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions

Abstract

Most chloroplast proteins are synthesized in the cytosol as preproteins with N-terminal cleavable transit peptides and are imported into the organelle through the TOC-TIC translocon system. Import involves a complex set of recognition and membrane translocation steps that ensure the fidelity and unidirectional transport of the polypeptide across the double-membrane chloroplast envelope. To understand the mechanism of import, the molecular interactions and energetics of each step must be defined. Here, we describe the methods for capturing intermediates in the import process through the manipulation of the energy state of chloroplasts, and the use of two different chemical cross-linking approaches to examine the molecular interactions that mediate the import process and to assess the assembly state of the translocons. These approaches can be employed to identify sequential protein-protein interactions, and thereby dissect the pathway and roles of import components during protein import into chloroplasts.

Fig. 1

GTP-dependent binding of preSSU to components of the TOC translocons. Energy-depleted, in vitro-translated [³⁵S]preSSU was incubated with isolated chloroplasts in the presence of 0.1 mM GTP to promote GTP-dependent binding. The homobifunctional cross-linker DTME was added to cross-link the bound preprotein to TOC components. The chloroplasts were detergent solubilized under denaturing conditions and proteins immunoprecipitated with Toc159 or Toc33 affinity-purified antibodies. The immunoprecipitates were analyzed by SDS-PAGE and phosphorimaging to quantify co-immunoprecipitated [³⁵S]preSSU or by immunoblotting to detect Toc159 or Toc33.

Fig. 2

Stabilization of the association of Hsp93 and Tic40 with TOC–TIC complexes. Detergent extracts from isolated chloroplasts that had been treated with DSP at the concentrations indicated (lanes 2–4) or DMSO as a control (lane 1) were subjected to immunoprecipitation with anti-Tic110 antibodies. The immunoprecipitates were immunoblotted with the antibodies indicated at the right of the figure.