Protein import into chloroplasts involves redox-regulated proteins.

Pre-protein translocation into chloroplasts is accomplished by two distinct translocation machineries in the outer and inner envelope, respectively. We have isolated the translocon at the inner envelope membrane (Tic complex) by blue-native PAGE and describe a new Tic subunit, Tic62. Tic62, together with Tic110 and Tic55, forms a core translocation unit. The N-terminus of Tic62 shows strong homologies to NAD(H) dehydrogenases in eukaryotes and to Ycf39-like proteins present in cyanobacteria and non-green algae. The stromal-facing C-terminus of Tic62 contains a novel, repetitive module that interacts with a ferredoxin-NAD(P)(+) oxidoreductase. Ferredoxin-NAD(P)(+) oxidoreductase catalyses the final electron transfer of oxygenic photosynthesis from ferredoxin to NAD(P). Substrates that interfere with either NAD binding, such as deamino-NAD, or influence the ratio of NAD(P)/NAD(P)H, such as ruthenium hexamine trichloride, modulate the import characteristics of leaf-specific ferredoxin-NAD(P)(+) oxidoreductase isologues differently. We conclude that the Tic complex can regulate protein import into chloroplasts by sensing and reacting to the redox state of the organelle.

A Toc75-like protein import channel is abundant in chloroplasts.

Chloroplasts import post-translationally most of their constituent polypeptides via two distinct translocon units located in the outer and inner envelope. The protein import channel of the translocon of the outer envelope of chloroplasts, Toc75, is the most abundant protein in that membrane. We identify a novel Toc75 homologous protein, atToc75-V, a prominent protein that is clearly localized in the chloroplastic outer envelope. Phylogenetic analysis indicates that Toc75-V is more closely related to its prokaryotic ancestors than to Toc75 from plants. The presence of a second translocation channel suggests that alternative, previously unrecognized import routes into chloroplasts might exist.

The preprotein conducting channel at the inner envelope membrane of plastids.

The preprotein translocation at the inner envelope membrane of chloroplasts so far involves five proteins: Tic110, Tic55, Tic40, Tic22 and Tic20. The molecular function of these proteins has not yet been established. Here, we demonstrate that Tic110 constitutes a central part of the preprotein translocation pore. Dependent on the presence of intact Tic110, radiolabelled preprotein specifically interacts with isolated inner envelope vesicles as well as with purified, recombinant Tic110 reconstituted into liposomes. Circular dichroism analysis reveals that Tic110 consists mainly of beta-sheets, a structure typically found in pore proteins. In planar lipid bilayers, recombinant Tic110 forms a cation-selective high-conductance channel with a calculated inner pore opening of 1.7 nm. Purified transit peptide causes strong flickering and a voltage-dependent block of the channel. Moreover, at the inner envelope membrane, a peptide-sensitive channel is described that shows properties basically identical to the channel formed by recombinant Tic110. We conclude that Tic110 has a distinct preprotein binding site and functions as a preprotein translocation pore at the inner envelope membrane.