The Chloroplast Protein Import Channel Toc75: Pore Properties and Interaction with Transit Peptides

Abstract

The channel properties of Toc75 (the protein import pore of the outer chloroplastic membrane) were further characterized by electrophysiological measurements in planar lipid bilayers. After improvement of the Toc75 reconstitution procedure the voltage dependence of the channel open probability resembled those observed for other beta-barrel pores. Studies concerning the pore size of the reconstituted Toc75 indicate the presence of a narrow restriction zone corresponding to the selectivity filter and a wider pore vestibule with diameters of approximately 14 A and 26 A, respectively. Interactions between Toc75 and different peptides (a genuine chloroplastic transit peptide, a synthetic peptide resembling a transit peptide, and a mitochondrial presequence) show that Toc75 itself is able to differentiate between these peptides and the recognition is based on both conformational and electrostatic interactions.

Functional and Phylogenetic Properties of the Pore-forming β-Barrel Transporters of the Omp85 Family

“…The Omp85s of this branch exhibit a conductivity of ϳ500 pS ( Figs. 2 and 3; Table 1) (15,18). In contrast, the E. coli YaeT, a member of the Omp85 family, and the D. melanogaster Sam50 have a conductivity of ϳ100 pS (Table 1), which again supports our phylogenetic analysis.…”

“…PTBs of recent interest are, e.g. outer membrane proteins (which secrete adhesins such as hemagglutinin) (8,9) and bacterial (1, 7, 10 -12), mitochondrial (Tob55/Sam50) (5,13,14), and chloroplast outer membrane proteins (Toc75) (15) of the Omp85 family. The PTBs are partitioned into two functional categories, namely in translocation of precursor proteins across the membrane and in the assembly of outer membrane proteins (3).…”

“…A precursor protein-binding site at Toc75 (15,18), together with the action of Toc159 (19), facilitates the translocation of precursor proteins across the membrane. In contrast, the Omp85 proteins from Neisseria meningitidis, Escherichia coli, and mitochondria are involved in the assembly of outer membrane proteins (1,5,7,(11)(12)(13)(14).…”

The Arabidopsisppi1Mutant Is Specifically Defective in the Expression, Chloroplast Import, and Accumulation of Photosynthetic Proteins[W]

“…Although their precise mode of action remains unclear (Sveshnikova et al, 2000;Hiltbrunner et al, 2001b), Toc159 and Toc34 interact with incident preproteins (Ma et al, 1996;Kouranov and Schnell, 1997) and mediate their transfer to the translocation channel, of which Toc75 is a major component. Toc75 has a ␤ -barrel structure comprising 16 amphiphilic ␤ -strands and forms a channel with a pore size of ‫ف‬ 14 to 26 Å (Hinnah et al, 2002). The Tic complex is less well characterized, and there is even some disagreement in the literature concerning the identity of its components (Jarvis and Soll, 2002).…”

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).…”