Crystal structure of a fusion protein revealing the interactions between CrRbcX-IIa and the C-terminal tail of CrRbcL.

<p>(A) Unbiased omit difference electron density for the RbcL tail residues of the CrRbcL(462–474)-RbcX-IIa(37–156) fusion protein. The C-terminal sequence of CrRbcL is shown as a coil and the sidechains in stick representation. The difference electron density at 1.5 σ level is shown as orange meshwork. CrRbcX-IIa(37–156) is represented as a molecular surface. (B) Detailed view of the RbcL-RbcX interactions. The area boxed in panel (A) is shown. (C) Superposition of the CrRbcX-IIa(37–156) onto the Syn6301-RbcL₈/AnaCA-RbcX₈ crystal structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135448#pone.0135448.ref010" target="_blank">10</a>]. The structures are shown in ribbon representation. The RbcL subunits are shown in brown and siena; the AnaCA-RbcX dimer in red; CrRbcX-IIa dimer in blue. (D) Putative contacts of CrRbcX-IIa(37–156) with the surface of the Syn6301-RbcL₈ complex. The same view as in panel (C) is shown.</p

Crystal structure of the CrRbcX-IIa(34–156) dimer.

<p>(A) Ribbon representation of the CrRbcX-IIa(34–156) dimer. Two perpendicular views are shown, the first along the molecular two-fold axis. (B) Interactions of the N-terminal tail with the hydrophobic cleft in CrRbcX-IIa(34–156). A zoom-in on the boxed area in panel (A) is shown. The N-terminal tail is shown as a coil with prominent sidechains in stick representation. The bulk of the CrRbcX-IIa(34–156) is represented as a molecular surface.</p

Sequence alignment of RbcX-II from green algae.

<p>Amino acid sequences of selected RbcX-II homologs from green algae, mosses and plants were aligned using Clustal-Ω. Note that for the green algae <i>Coccomyxa subellipsoidea</i>, <i>Chlorella variabilis</i>, <i>Volvox carteri</i>, <i>Ostreococcus tauri</i> and <i>Micromonas pusilla</i> only one RbcX-II sequence is shown. For comparison, RbcX-I from <i>A</i>. <i>thaliana</i>, <i>Synechococcus</i> sp. PCC7002 and <i>Anabaena sp</i>. CA are also aligned. All sequence numbering is based on the open reading frames. Secondary structure elements are indicated above the sequences. In the alignment, similar residues are shown in red and identical residues in white using bold lettering on red background. Blue frames indicate homologous regions. The consensus sequence is shown at the bottom. The forward arrow designates the beginning of the mature RbcX-II proteins. The diamond symbol at the end of the CrRbcX-IIb sequence indicates that the sequence continues with 130 amino acids not displayed. Asterisks denote residues known to be essential for RbcX function.</p

Oligomeric state of CrRbcX-IIa analyzed by native-MS.

<p>Nano-ESI native-MS spectra of CrRbcX-IIa(33–189) (A) and CrRbcX-IIa(34–156) (B). Symbols indicate the charge state distributions with the charge states shown for some peaks; the calculated mass around the <i>m/z</i> values of the respective protein complexes is reported. The accuracy of mass values calculated from the different <i>m/z</i> peaks is indicated.</p

Rubisco reconstitution of CrRbcX-IIa and oligomeric state of CrRbcX-IIa(34–189) analyzed by native-MS.

<p>(A) Rubisco reconstitution. Chemically denatured RbcL from <i>S</i>. <i>elongatus</i> PCC6301 (at 100 μM) was diluted 200-fold into ice-cold buffer containing GroEL (1.0 μM). The components (2 μM GroES oligomer; 2 μM AnaCa-RbcX or 30 μM CrRbcX dimer) were added as indicated and refolding/assembly initiated by addition of 4 mM ATP at 25°C (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135448#sec002" target="_blank">Materials and Methods</a>). After incubation for 60 min, RbcS (5 μM) was added with or without C-terminal RbcL peptide (200 μM) for 15 min, followed by Rubisco enzyme assay. The activity of RbcL₈ core complex (~0.06 μM oligomer) incubated with RbcS (5 μM) was set to 100%. Error bars s.d. (n = 3 independent experiments). (B) Nano-ESI native-MS spectra of CrRbcX-IIa(34–189). Symbols indicate the charge state distributions with the charge states shown for some peaks; the calculated mass around the <i>m/z</i> values of the respective protein complexes is reported. The accuracy of mass values calculated from the different <i>m/z</i> peaks is indicated.</p

Crystallographic data collection and model refinement statistics.

<p>* Values in parenthesis for outer shell.</p><p>** Values in parenthesis for test set.</p><p>*** Values from Molprobity 4.02.</p><p>Crystallographic data collection and model refinement statistics.</p

Regulation of phosphorylated eIF2α levels by inhibition of its phosphorylation and rescue of ST<i>Hdh</i>^Q111/111 cells.

<p><b>A</b>) EIF2α phosphorylation in ST<i>Hdh</i>^Q111/111 cells is PERK-mediated. ST<i>Hdh</i>^Q111/111 cells left untreated or treated with the PERK inhibitor A4 (50 µM) or the PKR inhibitor PKRi (1 µM) for the indicated times. **P = 0.009. <b>B</b>) ER stress-mediated eIF2α phosphorylation is inhibited by A4 and not by PKRi. As in (A), but with ST<i>Hdh</i>^Q7/7 cells treated for different times with Tun. <b>C</b>) PKR-mediated eIF2α phosphorylation is inhibited by PKRi and not by A4. As in (B), but with cells treated for 7h with the PKR inducer poly-I:C (200 µg/ml). <b>D-E</b>) A4 rescued ST<i>Hdh</i>^Q111/111 cells from UPR-induced cell death (Tun for 48 h, D), whereas PKRi had no effect (E). ***P =  0.0001. <b>F</b>) Total protein synthesis levels are much increased in ST<i>Hdh</i>^Q111/111 cells after prolonged ER stress (Tun for 24h) and reduced by A4 (50 µM). **P<0.002 (3 repeat experiments).</p

High sensitivity of striatal neurons to ER stress, further aggravated by expression of pathogenic huntingtin.

<p><b>A-C</b>) Strong induction of GADD34 and CHOP upon prolonged ER stress in ST<i>Hdh</i>^Q7/7 cells and even stronger in ST<i>Hdh</i>^Q111/111 cells; (3 independent experiments ±SE). *P = 0.02, <b>*</b>*P = 0.01, ***P = 0.0002. Immunoblots of a representative experiment are shown in A. GAPDH levels served here as a loading control. <b>D</b>) Prolonged ER stress induced with Tun or MG-132 leads to extensive death of ST<i>Hdh</i>^Q7/7 cells, further aggravated in ST<i>Hdh</i>^Q111/111 cells, as measured by FACS analysis of cell cycle progression with propidium iodide (PI) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090803#pone.0090803.s002" target="_blank">Fig. S2</a>); (6 independent experiments ± SE). *P<0.05, **P = 0.01, ***P = 0.001.</p

Regulation of phosphorylated eIF2α levels by inhibition of its dephosphorylation.

<p><b>A</b>) Guanabenz (Gz), at a relatively high concentration (100 µM), inhibits eIF2α dephosphorylation in untreated ST<i>Hdh</i>^Q7/7 cells and also in those treated with Tun (5 µg/ml) up to 7h; this is also true in ST<i>Hdh</i>^Q111/111 cells but only after very short treatments. *P = 0.02, **P = 0.01. EIF2α-P levels were normalized by total eIF2α. <b>B</b>) Similar to (A), but for cells treated for 24 h. After these long treatments Gz did not inhibit ER stress-induced eIF2α dephosphorylation, it increased CHOP levels. The values in the graphs are averages from 3-4 independent experiments±SE. *P<0.05, **P = 0.002. <b>C</b>) Gz showed a minor effect in rescuing ST<i>Hdh</i>^Q111/111 cells from UPR-induced cell death (Tun for 48 h). ***P =  0.0001.</p

Very low eIF2α-P levels in striatal cells, much increased by expression of Htt111Q.

<p><b>A</b>) Basal level of eIF2α-P in murine cell lines normalized by total eIF2α. Graph: average of 3 experiments ± SE<b>.</b> **P = 0.004, ***P  = 0.001. <b>B</b>) Immunofluorescence images of cells fixed, permeabilized and stained with rabbit anti-eIF2α-P and mouse anti-eIF2α followed by secondary antibodies. Bar = 10 µm. Image exposure time was kept constant to be able to compare protein levels in the different cell types. Levels relative to ST<i>Hdh</i>^Q7/7 levels were quantified from images from 3 experiments ± SE (>20 cells, ***P<0.001).</p