Research Article

Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2

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Science  08 Dec 2017:
Vol. 358, Issue 6368, pp. 1272-1278
DOI: 10.1126/science.aap9221
  • Fig. 1 Plant RuBisCo folding and assembly in E. coli requires coexpression of chloroplast chaperonin and auxiliary factors.

    (A) Operon organization of plasmids encoding A. thaliana RuBisCo (pAtRbcLS); chloroplast chaperonin factors (pAtC60αβ/C20); and predicted RuBisCo biogenesis factors Raf1, Raf2, RbcX, and BSD2 (pAtR1/R2/RX/B2) (fig. S1A). RBS, ribosome binding site. (B) Native-PAGE analysis of cell extracts from E. coli cells expressing AtRbcL and AtRbcS with and without auxiliary factors, as indicated (lanes 2 to 5). RuBisCo holoenzyme from A. thaliana leaf extract (lane 1) was used as standard. EV, empty vector control. Asterisk marks the position of chloroplast or E. coli chaperonins. (C) RuBisCo synthesized in A. thaliana leaves and in E. coli show equivalent carboxylation rates (Embedded Image). Data are averages ± SD from at least three independent experiments. (D) Analysis by means of SDS-PAGE of partially purified, recombinantly expressed AtRuBisCo. Impurities are marked with asterisks. The enzyme purified from leaves as well as recombinant AtRbcL and AtRbcS were used as standards (fig. S1B).

  • Fig. 2 Chaperonin dependence of AtRbcL folding.

    (A and B) AtRuBisCo production in E. coli strains expressing A. thaliana chloroplast chaperonin proteins AtCpn60α/β (AtC60α/β), AtCpn20 (C20), and AtCpn10 (C10) or the E. coli chaperonin system GroEL/GroES (EcEL/ES) in the combinations indicated. (A) Analysis of soluble cell lysates by means of native-PAGE and antibody-to-RbcL immunoblot. Native-PAGE samples were loaded on the basis of equal OD600 (optical density of a sample measured at a wavelength of 600 nm) of cells (~15 μg total protein per lane). Asterisk indicates chaperonin-bound RbcL. (B) RuBisCo activity in cell lysates through CO2 fixation (black bars) and densitometry of native-PAGE immunoblot to quantify RuBisCo amount (gray bars). Data are averages ± SD from three independent experiments.

  • Fig. 3 Chaperone dependence of AtRuBisCo assembly.

    (A to C) RuBisCo content analysis in E. coli strains upon deletion of specific auxiliary factors from pAtR1/R2/RX/B2 (lanes 2 to 6) or containing empty pCDF-Duet vector (lane 1) (fig. S2). (A) Native-PAGE and antibody-to-RbcL immunoblot. (B) RuBisCo content in soluble lysates through CO2 fixation (black bars) and [14C]-CABP binding (gray bars). Amounts of RuBisCo are expressed as percent of total soluble protein. Data are averages ± SD from three independent experiments. (C) Soluble expression of RbcL by means of SDS-PAGE and antibody-to-RbcL immunoblotting. T, total lysate fractions; S, soluble lysate fractions obtained by means of centrifugation. Equivalent amounts of fractions were analyzed. (D and E) Recombinant expression and assembly efficiency of N. tabacum RuBisCo in the presence of A. thaliana chaperones or substitution of N. tabacum Raf1. (D) NtRbcL and NtRbcS were expressed in E. coli with coexpression of the A. thaliana chaperonin system and auxiliary factors (lane 3) or upon substitution of AtRaf1 by NtRaf1 (lane 4). NtRuBisCo from tobacco leaves was analyzed as standard (lane 1), and AtRuBisCo was expressed as control (lane 2). Native- and SDS-PAGE samples were loaded with equal amounts of protein (20 and 12 μg per lane, respectively). (E) RuBisCo carboxylation activity was analyzed as above. Data are averages ± SD from three independent experiments.

  • Fig. 4 Functional role of A. thaliana BSD2.

    (A to C) Formation of three distinct high-molecular-weight RbcL complexes at RbcS limiting conditions (6% native PAGE at 200 V for ~1.5 hours). Top band (T), RbcL/BSD2; lower band (L), RbcL/BSD2/RbcS. (A) Coomassie-stained native-PAGE. (B) Native-PAGE immunoblots against RbcL, RbcS, and BSD2. Expression of AtRuBisCo with all auxiliary factors (lane 1), with additional RbcS (lane 2) or expression of RbcL in the absence of RbcS (lane 3). (C) Overlay of BSD2 and RbcS immunoblots, with RbcS in pink and BSD2 in cyan. The middle band (white) contains both BSD2 and RbcS. (D) Native-MS spectra of SeRbcL8 complexes incubated with AtBSD2 at increasing molar ratios (SeRbcL8:AtBSD2 1:0.5 to 1:2) for 15 min at 25°C. (E) Native-MS spectra of AtBSD2. Charge-state distributions are shown with corresponding symbols for each RbcL:BSD2 complex population. The calculated mass around the m/z values of the respective protein complexes and the accuracy of mass values calculated from the different m/z peaks are indicated. (F) Native-PAGE and immunoblot analysis of SeRbcL8 complexes alone (lane 1) and upon incubation for 30 min at 25°C with AtBSD2 (lane 2), SeRbcS (lane 3), or upon incubation with AtBSD2 for 15 min followed by addition of SeRbcS for 15 min (lane 4). The molar ratio of RbcL to BSD2 or RbcS was 1:2. Immunoblotting with antibodies to RbcL and BSD2. Relative CO2 fixation activities of the reactions are indicated. Data are averages ± SE from three independent experiments.

  • Fig. 5 Crystal structures of AtBSD2 and heterologous TeRbcL8:AtBSD28 complex.

    (A) Ribbon representation of the AtBSD2 crystal structure. Two perpendicular views are shown. The Zn centers and cysteine ligands are shown in space-filling and stick representation, respectively. The amino acid sequence of the crystallized AtBSD2 construct is shown schematically. Green, sequence resolved in the structure; residues 57 to 68 and 130 to 136 are unstructured. TP, transit peptide. (B) Surface properties of AtBSD2. Hydrophobic side chains are indicated in yellow. Red and blue represent negatively and positively charged groups, respectively. (C) Surface conservation of AtBSD2. Color gradient from cyan to magenta represents increasing conservation, based on sequence alignment of BSD2 homologs (fig. S4B). The positions of residues chosen for mutational analysis are indicated. (D) Crystal structure of the TeRbcL(IA)8:AtBSD28 complex. BSD2 (green) is shown in ribbon representation. RbcL8 is shown as surface with RbcL in white and RbcL’ in light orange. (E) Interactions between Zn center 2 of BSD2 (green) and RbcL (white). Critical amino acid residues of BSD2 and RbcL are shown in red and yellow stick representation, respectively. (F) Interactions between Zn center 1 of BSD2 (green) and the RbcL2 unit. Interacting residues are colored as in (E). (G) Rearrangement of the 60s loop in TeRbcL(IA)8:AtBSD28 complex (green) compared with apo-RuBisCo (DOI: 10.2210/pdb2ybv/pdb) and CABP-bound RuBisCo (DOI: 10.2210/pdb3zxw/pdb) (red and blue, respectively). RbcS and AtBSD2 are shown for orientation. (H) Interaction of the C-terminal tail of BSD2 with the catalytic center of RbcL2. BSD2 in green and the RbcL in the RbcL8:BSD28 complex in white/light orange ribbon representation. The position of loop 6 in the CABP-bound RuBisCo is shown in cyan.

  • Fig. 6 Function of auxiliary factors in RuBisCo assembly.

    (A to C) Mutational analysis of AtBSD2. (A) Soluble expression of mutant proteins. Wild-type (WT)–AtBDS2 in pR1/R2/RX/B2 was replaced with the mutant proteins indicated. Cell extracts were fractionated as in Fig. 3C, and total (T) and soluble (S) fractions were analyzed by means of antibody-to-AtBSD2 immunoblotting. (B) Native-PAGE and immunoblot analysis of RuBisCo assembly in E. coli cells expressing mutant AtBSD2 (lanes 4 to 9). Cells lacking AtBSD2 (lane 2) or expressing WT-AtBSD2 (lane 3) served as control. Purified AtRuBisCo from leaf extracts is shown in lane 1. (C) Relative RuBisCo carboxylation activities in cell extracts as above. Data are averages ± SE from three independent experiments. (D) Model of chaperone-assisted folding and assembly of plant RuBisCo. Upon folding of newly synthesized RbcL subunits by the Cpn60αβ/Cpn20 chaperonin, RbcL assembly to RbcL dimers and higher oligomers is mediated by Raf1 and RbcX acting in cooperation or in parallel. Binding of BSD2 causes the displacement of these factors and stabilizes RbcL8 cores in a state competent for association with RbcS. RbcS binding causes displacement of BSD2, forming the functional holoenzyme. Raf2 is essential for RuBisCo biogenesis and may act downstream or upstream of chaperonin.

Supplementary Materials

  • Plant Rubisco assembly in E. coli with five chloroplast chaperones including BSD2

    H. Aigner, R. H. Wilson, A. Bracher, L. Calisse, J. Y. Bhat, F. U. Hartl, M. Hayer-Hartl

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods 
    • Figs. S1 to S5 
    • Tables S1 to S3 
    • References

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