Journal: The Plant Journal

Article Title: Structural characterizations of the chloroplast translocon protein Tic110

doi: 10.1111/tpj.12249

Figure Lengend Snippet: Purification of the three CmTic110 proteins. (a) Schematic representations of the two structural models of Tic110 and the corresponding regions covered by CmTic110 A , CmTic110 B and CmTic110 C . Locations of the proposed transmembrane domains (TM1 and TM2 are represented by yellow boxes; TM3 to TM6 are represented by orange boxes), transit peptides (TP, pink boxes) and domains for transit-peptide binding (green box) and (co) chaperone binding (purple box) are marked. (b) CmTic110 proteins were purified to homogeneity. One microgram of purified proteins was analyzed by SDS-PAGE and stained by Coomassie blue. (c) Purified CmTic110 proteins were analyzed by gel filtration. The chromatograph of each CmTic110 protein is shown.

Article Snippet: [ 35 S]Met-CmTic110 and CmTic110D363x were in vitro transcribed/translated in the TNT reticulocyte lysate system (Promega).

Techniques: Purification, Binding Assay, SDS Page, Staining, Filtration

Journal: The Plant Journal

Article Title: Structural characterizations of the chloroplast translocon protein Tic110

doi: 10.1111/tpj.12249

Figure Lengend Snippet: Sequence alignment and domain features of Tic110s. Tic110 homologues from Pisum sativum (Ps), Arabidopsis thaliana (At), Physcomitrella patens (Pp), Chlamydomonas reinhardtii (Cr) and Cyanidioschyzon merolae (Cm) were aligned by clustal W2, generated from ESPript (Gouet et al ., ) and further manually adjusted. Fully conserved residues are shaded in red and regions with similar residues are boxed in blue. The transit-peptide processing site in PsTic110 is indicated by a downward arrow. The transmembrane helices are shaded in yellow and orange. The region corresponding to the transit-peptide-binding domain of model 2 is boxed in green. The N termini of CmTic110 A , CmTic110 B and CmTic110 C are marked with rightward arrows. The C terminus of CmTic110D363x is marked with a leftward arrow. The conserved leucine-zipper-like motif (L-x-x-L-x-x-x-L-G) is indicated above the sequence.

Article Snippet: [ 35 S]Met-CmTic110 and CmTic110D363x were in vitro transcribed/translated in the TNT reticulocyte lysate system (Promega).

Techniques: Sequencing, Generated, Binding Assay

Journal: The Plant Journal

Article Title: Structural characterizations of the chloroplast translocon protein Tic110

doi: 10.1111/tpj.12249

Figure Lengend Snippet: CmTic110 is imported into pea chloroplasts. (a) In vitro translated [ 35 S]Met-CmTic110 (Tr, lane 1) was treated with thermolysin directly (lane 2) or incubated with isolated pea chloroplasts (Cpt, 100 μg chlorophyll) under import conditions for 30 min. After import, a small portion of the chloroplasts (10 μg chlorophyll) were centrifuged through a 40% Percoll cushion to re-isolate intact chloroplasts (lane 3). The rest of the chloroplasts were digested with thermolysin, and then recovered through a 40% Percoll cushion (lane 4). Some of the thermolysin-treated chloroplasts (81 μg chlorophyll) were further lysed hypotonically and separated into membrane (M, lane 5) and soluble (S, lane 6) fractions by centrifugation. Samples were analyzed by SDS-PAGE, Coomassie blue staining and fluorography. Twenty micrograms of proteins were loaded in lanes 3–6. The arrow indicates the full-length CmTic110 and the bracket marks the three imported and process CmTic110 inside chloroplasts. LS, large subunit of ribulose biphosphate carboxylase in the stroma, which serves as a marker for the soluble fraction; CAB, chlorophyll a / b -binding protein of the thylakoid membrane, which serves as a marker for the membrane fraction. (b) Import of CmTic110D363x. The experimental conditions and lane designations are the same as (a), except lane 5 is the soluble fraction and lane 6 is the membrane fraction.

Article Snippet: [ 35 S]Met-CmTic110 and CmTic110D363x were in vitro transcribed/translated in the TNT reticulocyte lysate system (Promega).

Techniques: In Vitro, Incubation, Isolation, Centrifugation, SDS Page, Staining, Marker, Binding Assay

Journal: The Plant Journal

Article Title: Structural characterizations of the chloroplast translocon protein Tic110

doi: 10.1111/tpj.12249

Figure Lengend Snippet: Small-angle X-ray scattering (SAXS) characterizations of CmTic110 B and CmTic110 C . (a, b) Experimental X-ray scattering curves (black line) and the theoretical fitting curves (red line) of CmTic110 B and CmTic110 C were generated with gnom . The insets show the Guinier plots. (c, d) The distance distributions of CmTic110 B and CmTic110 C . (e,f) Averaged low-resolution envelope obtained from 10 independent ab initio models of CmTic110 B and CmTic110 C derived from the SAXS data.

Article Snippet: [ 35 S]Met-CmTic110 and CmTic110D363x were in vitro transcribed/translated in the TNT reticulocyte lysate system (Promega).

Techniques: Generated, Derivative Assay

Journal: The Plant Journal

Article Title: Structural characterizations of the chloroplast translocon protein Tic110

doi: 10.1111/tpj.12249

Figure Lengend Snippet: Structure of CmTic110 C . (a) Electron density map of experimental phase at 1.5σ and model building of CmTic110 C based on the six selenium sites (red balls 1–6 for selenium in Met917, Met940, Met1017, Met1020, Met1051 and Met1212, respectively). (b) A ribbon drawing of the CmTic110 C structure. Helix 3 and part of the loop connecting helix 4 (indicated in yellow) is the corresponding region of TM6 in model 1 shown in Figure (a). (c) A close-up view of the region corresponding to TM6 and its dimensions. All the figures were generated with pymol ( http://www.pymol.org/ ).

Article Snippet: [ 35 S]Met-CmTic110 and CmTic110D363x were in vitro transcribed/translated in the TNT reticulocyte lysate system (Promega).

Techniques: Generated