Semi-quantitative analysis of Tic20 and Tic110 on protein level was performed using immunoblots of envelope membranes isolated from two-week-old pea and four week-old Arabidopsis plants. In parallel, calibration curves were generated using a series of known concentrations of over expressed and purified proteins . After quantification of immunoblots from envelopes, amounts of PsTic20, PsTic110, AtTic20 and AtTic110 were determined using the corresponding calibration curve. The amount of PsTic110 in IE was found to be almost eight times higher than that of PsTic20 , which differs strikingly from the similar transcript levels of the two genes detected in leaves , indicating profound differences in post translational processes such as translation rate and protein turnover. In Arabidopsis, the absolute amount of AtTic110 is nearly the same as in pea , however, Arabidopsis envelopes represent a mixture, containing both outer and IE vesicles. Thus, the relative amount of AtTic110 is possibly higher than in pea. Surprisingly, the amount of AtTic20 is more than 100 times lower than that of AtTic110, showing an even greater difference in comparison to the observed RNA expression levels . Taking the different molecular size of Tic110 and Tic20 into account , we still observe 20 times more AtTic110 than AtTic20 protein. In pea, we found 1.4 times more Tic110 RNA than Tic20,cultivo del arandano whereas in Arabidopsis the ratio of Tic110 to Tic20 is 20.3.

The number of channel forming units must even be more different, since Tic110 was shown to form dimers, whereas Tic20 builds very large complexes between 700 kDa and 1 MDa. Thus, two Tic110 molecules would be necessary to form a channel in contrast to Tic20, which would require many more molecules to form the pore. Though we cannot exclude that Tic20 might be subject to degradation by an unknown protease in vivo, protease treatments with thermolysin of right-side out IE vesicles in vitro clearly shows that Tic20 is very protease resistant,even in the presence of detergent. In contrast, Tic110 is easily degraded already without addition of detergent . This argues against more rapid degradation of Tic20 compared to Tic110 during preparation of IE. The difference in Tic110 to Tic20 ratios both on the RNA and protein level between pea and Arabidopsis may be due to the different age of the plants or the different needs under the given growth conditions, and suggests that there is no strict stoichiometry between the two proteins. Moreover, the low abundance of Tic20 in comparison to Tic110 in envelopes clearly demonstrates thatTic20 cannot be the main channel of the Tic translocon as previously suggested , since it cannot possibly support the required import rates of some highly abundant preproteins that are needed in the chloroplast.Experimental data suggested a common complex between Tic110 and Tic20 in chloroplast envelope membranes using a cross-linking approach. However, the interaction was not visible in the absence of Toc components, making a stable association unlikely.

Furthermore, no evidence for a common complex was found by Kikuchi et al. using solubilized chloroplasts of pea and Arabidopsis for two-dimensional blue native/SDS-PAGE analysis. Likewise, the difference in Tic110 to Tic20 ratios both on the RNA and protein level between pea and Arabidopsis indicates that a common complex, in which both proteins cooperate in translocation channel formation in a reasonable stoichiometry, is improbable. To clarify this issue, we addressed these partly conflicting results by using IE vesicles, which should minimize the possible influence of the interaction with Toc components on complex formation.Immunoblots revealed that both Tic20 and Tic110 are present in distinct high molecular weight complexes : Tic110-containing complexes migrate at a size of ~ 200-300 kDa, whereas Tic20 displays a much slower mobility in BN-PAGE and is present in complexes exceeding 700 kDa, in line with the results from Kikuchi et al.. However, at a similar molecular weight of 250 kDa on BN-PAGE not only Tic110 but also Hsp93, Tic62 and Tic55 were described . The molecular weight of a complex containing all of these components would be much higher. Therefore, components of the Tic complex might associate with Tic110 very dynamically resulting in different compositions under different conditions, or alternatively, there are different complexes present at the same molecular weight. An open question to date is the identity of possible interaction partners of Tic20 in the complex. Tic22, the only Tic component located in the intermembrane space, is a potential candidate, since both proteins were identified together in cross-linking experiments. However, only minor amounts of Tic20 and Tic22 were shown to co-localize after gel filtration of solubilized envelope membranes. A second candidate for common complex formation is PIC1/Tic21: Kikuchi et al. demonstrated that a one-megadalton complex of Tic20 contains PIC1/Tic21 as a minor subunit.

PIC1/ Tic21 was proposed to form a protein translocation channel in the Tic complex, mainly based on protein import deffects of knockout mutants and on structural similarities to amino acid transporters and sugar permeases. An independent study by Duy et al. favours the hypothesis that PIC1/Tic21 forms a metal permease in the IE of chloroplasts, rendering the import-related role questionable. This discrepancy will have to be addressed in the future. To test the complex formation of Tic20 in vitro without the involvement of other proteins, we used Tic20- proteoliposomes for 2D BN/SDS-PAGE analysis, similarly to IE vesicles . The migration behaviour of the protein resembles that observed in IE: the majority of the protein localizes in high molecular weight range, however, the signal appears more widespread and a portion is also detected at lower molecular weights, possibly as monomers. This observation reveals that Tic20 has the inherent ability to homo-oligomerize in the presence of a lipid bilayer. The less distinct signal could be due to different solubilization of Tic20 by digitonin in IE vesicles vs. liposomes, or could be an indication that additional subunits stabilize the endogenous Tic20 complexes, which are not present after the reconstitution. However, we interpret these observations as support for the hypothesis that the major component of the one megadalton complex in IE are homo-oligomers composed of Tic20.In silico analysis of Tic20 predicts the presence of four hydrophobic transmembrane helices positioning both N- and C-termini to one side of the membrane . According to these predictions, three cysteins in PsTic20 face the same side, while the fourth would be located in the plane of the membrane. We used pea IE vesicles prepared in a right-side-out orientation to determine the topology of Tic20 employing a Cys-labelling technique. To this end, the IE vesicles were incubated with a membrane-impermeable, Cys-reactive agent that adds a molecular weight of 5,000 Da to the target protein for each reactive Cys residue. In our experiments PEG-Mal did not strongly label any Cys residues of Tic20 under the conditions applied , indicating the absence of accessible Cys residues on the outside of the membrane. Only one faint additional band of higher molecular weight was detectable ,maceta hidroponica possibly due to a partially accessible Cys located within the membrane. In the presence of 1% SDS, however, all four Cys residues present in PsTic20 are rapidly PEGylated, as demonstrated by the appearance of four intense additional bands after only five minutes of incubation. The observed gain in molecular weight per modification is bigger than the expected 5 kDa for each Cys, but this can be attributed to an aberrant mobility of the modified protein in the Bis-Tris/ SDS-PAGE used in the assay. Our results support a four transmembrane helix topology in which both the C- and N-termini are facing the stromal side of the membrane , with no Cys residues oriented towards the intermembrane space. Cys108 is most likely located in helix one, Cys227 and Cys230 are oriented to the stromal side of helix four and Cys243 is located in the stroma. This topology is also in line with green fluorescent protein-labelling studies by van Dooren et al. indicating that the N- and C-termini also of the Toxoplasma gondii homolog of Tic20 face the stromal side of the inner apicoplast membrane.

Tic20 was identified more than a decade ago but since then no heterologous expression and purification procedure has been reported, which could successfully synthesize folded full-length Tic20. Here, we report two efficient Escherichia coli based systems for Tic20 expression and purification from both pea and Arabidopsis: codon optimized PsTic20 was over expressed in a S12 cell lysate in presence of detergents, and AtTic20 over expression was successfully accomplished by adaptation of a special induction system. Following these steps, both pea and Arabidopsis proteins could be purified to homogeneity by metal affinity purification . Using the purified protein, we performed structural characterization studies of Tic20 by subjecting it to circular dichroism spectroscopy . The recorded spectra of PsTic20, displaying two minima at 210 and 222 nm and a large peak of positive ellipticity centered at 193 nm, are highly characteristic of a-helical proteins, and thus demonstrate that the protein exists in a folded state after purification in the presence of detergent. The secondary structure of Tic20 was estimated by fitting spectra to reference data sets resulting in an a-helical content of approximately 78%, confirming in silico predictions.To better characterize Tic20 in a membrane-mimicking environment, heterologously expressed and purified AtTic20 was reconstituted into liposomes in vitro. Initially, flotation experiments were performed to verify a stable insertion. In the presence or absence of liposomes, Tic20 was placed at the bottom of a gradient ranging from 1.6 M to 0.1 M sucrose. In the presence of liposomes, Tic20 migrated to the middle of the gradient, indicating a change in its density caused by interaction with liposomes. In contrast, the protein alone remained at the bottom of the gradient . Proteoliposomes were also treated with various buffers before flotation , to test whether the protein is firmly inserted into the liposomalmembrane or just loosely bound to the vesicle surface. None of the applied conditions changed the migration behaviour of Tic20 in the gradient , indicating that Tic20 was deeply inserted into the liposomal membrane. Thus, proteoliposomes represent a suitable in vitro system for the analysis of Tic20 channel activity.Even though Tic20 has long been suggested to form a channel in the IE membrane, this notion was solely based on structural analogy to other four-transmembrane helix proteins, and no experimental evidence has been provided so far. To investigate whether Tic20 can indeed form an ion channel, Tic20-proteoliposomes were subjected to swelling assays . Changes in the size of liposomes in the presence of high salt concentrations, as revealed by changes in the optical density, can be used to detect the presence of a poreforming protein. After addition of 300 mM KCl to liposomes and Tic20-proteoliposomes, their optical densities dropped initially, due to shrinkage caused by the increased salt concentration. However, the optical density of protein-free liposomes remained at this low level, showing no change in their size; whereas in the case of Tic20-proteoliposomes the optical density increased constantly with time. The increase in optical density strongly supports the presence of a channel in Tic20-proteoliposomes that is permeable for ions, thereby creating an equilibrium between the inner compartment of the proteoliposomes and the surrounding buffer. To exclude the possible effects of contaminating channel-forming proteins derived from the bacterial membrane and a protein inserted into the liposomes , a further negative control was set up: Tic110 containing only the first three transmembrane helices was purified similarly to Tic20 and reconstituted into liposomes. We chose this construct, since NtTic110 inserts into the membrane during in vitro protein import experiments. Furthermore, as the full length and N-terminally truncated Tic110 possess very similar channel activities, it is unlikely that the N-terminal part alone forms a channel. The insertion of NtTic110 into liposomes was confirmed by incubation under different buffer conditions followed by flotation experiments, similarly to Tic20 . However, these NtTic110-proteoliposomes behaved similarly to the empty liposomes during swelling assays: after addition of salt, the optical density decreased, and except for a small initial increase, it remained at a constant level . This makes it unlikely that a contamination from E. coli or simply the insertion of a protein into the liposomes caused the observed effect in the optical density of Tic20- proteoliposomes. To further characterize the channel activity of Tic20, electrophysiological measurements were performed.