Reconstitution of a chloroplast protein import channel

Abstract

The chloroplastic outer envelope protein OEP75 with a molecular weight of 75 kDa probably forms the central pore of the protein import machinery of the outer chloroplastic membrane. Patch-clamp analysis shows that heterologously expressed, purified and reconstituted OEP75 constitutes a voltage-gated ion channel with a unit conductance of Λ ϭ 145pS. Activation of the OEP75 channel in vitro is completely dependent on the magnitude and direction of the voltage gradient. Therefore, movements of protein charges of parts of OEP75 in the membrane electric field are required either for pore formation or its opening. In the presence of precursor protein from only one side of the bilayer, strong flickering and partial closing of the channel was observed, indicating a specific interaction of the precursor with OEP75. The comparatively low ionic conductance of OEP75 is compatible with a rather narrow aqueous pore (d pore ≅ 8-9 Å). Provided that protein and ion translocation occur through the same pore, this implies that the environment of the polypeptide during the transit is mainly hydrophilic and that protein translocation requires almost complete unfolding of the precursor.

The Roles of Toc34 and Toc75 in Targeting the Toc159 Preprotein Receptor to Chloroplasts

“…For this purpose, we took advantage of a modified protocol for the reconstitution of functional psToc34 and psToc75 into lipid vesicles. Previous studies using similar procedures have demonstrated that the refolding of psToc34 and psToc75 into proteoliposomes results in the reconstitution of native activities (10,25). Both psToc75 and psToc34 proteins were expressed in E. coli and purified to near homogeneity under denaturing conditions in the presence of urea (data not shown).…”

“…Reconstitution of Proteoliposomes-Liposomes were generated using azolectin (type IV-S, Sigma) and a method adapted from Hinnah et al (10). Azolectin was resuspended in 10 mM MOPS-KOH, pH 6.9, to give a final concentration of 50 mg/ml and pulse sonicated for 30 s to facilitate even dispersion.…”

AtToc90, a New GTP-Binding Component of the Arabidopsis Chloroplast Protein Import Machinery

“…An alternative model in which Toc34 functions as the primary receptor and Toc159 as a GTP-dependent translocation motor has also been proposed (Schleiff et al, 2003). Toc75, the third component, forms at least part of a hydrophilic channel through which precursors are translocated across the outer membrane Hinnah et al, 1997;Hinnah et al, 2002).…”

The Omp85-Related Chloroplast Outer Envelope Protein OEP80 Is Essential for Viability in Arabidopsis

“…It associates with precursor proteins in vitro (Perry and Keegstra, 1994;Schnell et al, 1994), and was reconstituted as a cationselective ion channel in artificial liposomes (Hinnah et al, 1997), suggesting that it forms a major component of the preprotein translocation channel (Bédard and Jarvis, 2005;Kessler and Schnell, 2006;Smith, 2006). Unlike other outer membrane proteins, Toc75 is synthesized as a larger precursor with a bipartite targeting signal (Tranel et al, 1995;Tranel and Keegstra, 1996); the first part is a standard transit peptide for chloroplast import (Inoue et al, 2001) and the second part acts as an intraorganellar targeting signal that is cleaved by an envelope-bound type I signal peptidase (Inoue and Keegstra, 2003;Inoue et al, 2005;Baldwin and Inoue, 2006).…”