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. 2002 Jun 3;21(11):2616-25.
doi: 10.1093/emboj/21.11.2616.

The preprotein conducting channel at the inner envelope membrane of plastids

Lisa Heins  1 , Alexander Mehrle, Roland Hemmler, Richard Wagner, Michael Küchler, Friederike Hörmann, Dmitry Sveshnikov, Jürgen Soll
Affiliations

The preprotein conducting channel at the inner envelope membrane of plastids

Lisa Heins et al. EMBO J. .

Abstract

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.

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Figures

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Fig. 1. Binding of preprotein to isolated inner envelope vesicles. (A) Inner envelope vesicles (5 µg protein), the 35S-labelled small subunit of the ribulose bisphosphate carboxylase (SSU) and the light harvesting complex protein II (LHCP) were incubated. Only the preproteins (pSSU, pLHCP), not the mature forms (mSSU, mLHCP), were re-isolated together with inner envelope vesicles, i.e. 20% (SSU) or 10% (LHCP) of the translation product used for binding is shown in the left lane (–). An X-ray film is shown. (B) Purity of isolated inner envelope vesicles. Outer envelope vesicles (oe), inner envelope vesicles (ie) and thylakoids (thy) equivalent to 0.5 µg protein, respectively, were subjected to immunodecoration. Antisera raised against marker proteins of the outer envelope (Toc86, Toc75, Toc34 and OEP24), the inner envelope (Tic110) and the thylakoids (LHCP) were used. (C) Binding of preproteins is dependent on proteins at the inner envelope. Treatment of inner envelope vesicles with trypsin (Try) prior to binding decreased the binding of pSSU, pLHCP, the preprotein of ferredoxin (pFd) and the 23 kDa protein of the oxygen-evolving complex (pOE23). The left lane comprises 20% translation product. An X-ray film is shown. (D) Tic110 and Tic40 are protease-accessible at inner envelope vesicles. A ratio of 25 ng trypsin/µg inner envelope protein was used. The samples were subjected to SDS–PAGE, transferred to nitrocellulose and entire lanes were incubated with antisera raised against (α) Tic110, Tic22, Tic55, and Tic40. The inner envelope vesicles (8%) used for the binding assay with (+) or without (–) trypsin treatment were analysed. (E) Binding of pSSU to Tic110. Inner envelope vesicles were incubated with radiolabelled pSSU prior to cross-linking with sodium tetrathionate. After solubilization with SDS, the proteins were immunoprecipitated with antiserum raised against Tic110. The cross-linked product was cleaved with β-mercaptoethanol. An aliquot of each fraction (T, FT, W, E) was subjected to SDS–PAGE and analysed by autoradiography.
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Fig. 2. Reconstitution of Tic110 into liposomes. (A) Tic110 and the mutant proteins (ΔN, ΔC) containing an N- or C-terminal poly(His) tag, respectively, were reconstituted into liposomes. The purity of the proteins and the efficiency of reconstitution was finally examined by 25% High-Tris–Urea PAGE. As a control, inner envelope vesicles (i.e. 10 µg protein) were subjected to electrophoresis. A Coomassie Blue-stained gel is shown. The masses of the molecular weight standards are given in kDa at the left side. (B) Protease treatment of inner envelope vesicles and Tic110 liposomes. Inner envelope vesicles (i.e. 2.5 µg protein) and Tic110 liposomes (25 ng protein) were treated with 25 ng trypsin (Try)/µg protein. After SDS–PAGE (10% acrylamide), the protein was transferred to nitrocellulose. The proteolytic pattern was detected with antiserum against (α) Tic110. Characteristic bands at 70 kDa and a doublet at ∼45 kDa are indicated by asterisks. The masses of the molecular weight standards are given in kDa at the left side.
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Fig. 3. Binding of preprotein to Tic110 and mutant proteins reconstituted into liposomes. (A) After binding, 35S-labelled pSSU was re-isolated together with inner envelope vesicles (5 µg protein), Tic110-proteoliposomes and ΔN-proteoliposomes (50 ng protein), but was not recovered together with ΔC-proteoliposomes. After binding and re-isolation, half of each sample was treated with thermolysin (Thl). The translation product (20%) used for binding is shown in the left lane (–). (B) As a control, the same experiment was performed with liposomes that did not contain protein, but were treated like proteoliposomes. The translation product (20%) used for binding is shown in the left lane (–). Binding was examined by 25% High-Tris–Urea PAGE. X-ray films are shown.
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Fig. 4. Tic110 forms a cation-selective channel sensitive to a chloroplast transit peptide. (A) Based on a circular dichroism spectra of Tic110 solved in 1% Mega-9, 50 mM KiPO4 pH 7.2, the relative abundance of secondary structures was calculated using a neural network (Dalmas et al., 1994). (B) Current traces from a bilayer containing multiple copies of the channel formed by recombinant Tic110, at different membrane potentials with 250 mM KCl, 2 mM CaCl2 and 10 mM MOPS–Tris pH 7.2 at both sides of the membrane. Subconductance levels were rarely observed. The value of the membrane potential applied is indicated at the right side. (C) Current–voltage relationship of a fully open single channel. The trans and the cis chamber symmetrically contain 250 mM KCl. The values were determined on average of four independent bilayers. The slope conductance of Λ = 446 ± 9 pS indicates a high conductance channel. (D) Ion selectivity of the Tic110 channel. In response to a voltage gradient starting at zero to a positive holding potential, the permeation of cations was preferred over anions. With asymmetric buffers at both sides of the bilayer (250 mM KCl cis/20 mM KCl trans) the channel had a reversal potential of Erev = 44 ± 1.6 mV (on average of 12 independent bilayers). (E) The inner diameter of the Tic110 channel. The conductance of the Tic110 channel in the presence of differently sized non-electrolyte PEG molecules was measured. The values of the narrowest part (filter) and the widest part (vestibule) of the pore were estimated. The hydronamic radius of the PEG molecules versus the quotient of the conductance in the presence of the non-electrolyte PEG (condPEG) and the conductance without PEG (cond0) is shown. Dashed lines indicate the narrowest and the widest opening. (F) The Tic110 channel is sensitive to chloroplast transit peptide. Purified transit peptide (trOE33) was added at the trans side (250 mM KCl) of the bilayer containing Tic110 and stirred for 10 s. Next, a voltage sweep (Δ = 10 mV/s) was applied and a block of the Tic110 channel starting at a membrane potential of ∼70 mV was observed (black). In the absence of trOE33, a linear current–voltage relationship occurred (gray). The activity of a bilayer containing five channels is shown.
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Fig. 5. Characterization of a Tic110 ΔN mutant. (A) Circular dichroism spectrum of the ΔN mutant Tic110 solved in 2% N-decyl-β-d-maltopyranoside and 50 mM KiPO4 pH 7.2. (B) Current traces from a bilayer containing multiple copies of the channel formed by the ΔN mutant Tic110. At both sides of the membrane, 250 mM KCl, 2 mM CaCl2 and 10 mM MOPS–Tris pH 7.2 were present. With different positive and negative membrane potentials, subconductance levels of the ΔN mutant channel were rarely observed. (C) Current–voltage relationship of a fully open single ΔN mutant channel. The trans and the cis chamber symmetrically contain 250 mM KCl. The values were determined on average of five independent bilayers. The slope conductance of 520 ± 10 pS indicates a high conductance channel.
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Fig. 6. Inner envelope vesicles contain a channel with properties identical to recombinant Tic110. (A) Current traces from a bilayer containing a single Tic110-like channel at different membrane potentials with 250 mM KCl, 2 mM CaCl2 and 10 mM MOPS–Tris pH 7.2 at both sides of the membrane. The value of the membrane potential is indicated at the right side. (B) Current–voltage relationship of a fully open single channel. The trans and the cis chamber symmetrically contain 250 mM KCl. The values are shown on average of three independent bilayers. The slope conductance of Λ = 600 ± 8 pS indicates a high conductance channel. (C) The channel exhibits cation selectivity. Current recordings obtained from a bilayer containing multiple copies of active Tic110 channels in response to a voltage ramp are shown. With asymmetric buffers (250 mM KCl cis/20 mM KCl trans), the reversal potential of 34 ± 1.6 mV was deduced, indicating that the channel is cation-selective. (D) The cation-selective Tic110-like channel at the inner envelope membrane is sensitive to chloroplast transit peptide. With 250 mM KCl cis and 20 mM KCl trans, a voltage sweep (Δ = 10 mV/s) was applied to the membrane and a linear correlation was observed (gray). After addition of transit peptide (trOE33) at the cis side, at a membrane potential of ∼45 mV, the current passing through the channel significantly decreased (black). The activity of a bilayer containing three channels is shown.
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Fig. 7. Model of the Tic complex. Tic110 forms at least a major part of the preprotein translocation pore. At the same time Tic110 might be involved in the formation of joint translocation sites together with the Toc complex and in recruiting of stromal chaperones (cpn60, Hsp100). The numbers indicate the calculated molecular weight of the Toc and Tic components.
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