Schematic depiction of equilibria between various co-chaperonin species of <i>A. thaliana</i>.

<p>Key: Cpn10(1) subunit - purple, Cpn10(2) subunit - green, Cpn20 subunit - orange (N-terminus) and yellow (C-terminus).</p

Effect of Cpn10(1) on co-chaperonin activity of Cpn20.

<p>MDH refolding was carried out by either (A) GroEL or (B) Cpn60αβ, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113835#s2" target="_blank">Materials and Methods</a>, in the presence of increasing concentrations of various co-chaperonin species, as indicated. 100% was taken as the activity of GroEL assisted by a saturating concentration of GroES, or the activity of Cpn60αβ assisted by mt-cpn10. Results are presented as an average of 3 independent experiments ± SD.</p

Functional synergism between inactive co-chaperonin species Cpn10(1) and G32A.

<p>MDH refolding was carried out by either (A) GroEL or (B) Cpn60αβ, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113835#s2" target="_blank">Materials and Methods</a>, in the presence of increasing concentrations of various inactive co-chaperonin species, as indicated. 100% was taken as the activity of GroEL assisted by a saturating concentration of GroES, or the activity of Cpn60αβ assisted by mt-cpn10. Results are presented as an average of 3 independent experiments ± SD.</p

Analysis of hetero-oligomer formation by gel filtration.

<p>Elution profile of ∼1.5 mg Cpn20 (top panels), ∼1.5 mg Cpn10(1) (bottom panels) or a mixture of the two (middle panels), separated by gel filtration on a Superdex 75 column at 4°C at a flow rate of 1 mg/ml. Fractions were subsequently analyzed by either SDS (left panel) or native (right panel) PAGE (20 µl per lane). Cpn20 and Cpn10(1) are indicated for each gel. The hetero-oligomeric species is marked with an arrow on the native gel. Fractions 14–17 contain primarily 70–80 kDa species while fractions 20–23 contain primarily 10–20 kDa species.</p

Effect of various co-chaperonins on the reconstitution of Cpn60β1 oligomers.

<p>Cpn60β1 oligomer was assembled from monomeric form as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113835#s2" target="_blank">Materials and Methods</a>, in the presence of Mg-ATP and various co-chaperonins or co-chaperonin mixtures as indicated. 3 µl of reconstitution reaction was loaded on a 6% native polyacrylamide gel. The reconstituted oligomers are indicated by an asterisk. GroEL oligomer is presented as a control.</p

Effect of G32A on co-chaperonin activity of Cpn10(2).

<p>MDH refolding by either A) GroEL or B) Cpn60αβ was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113835#s2" target="_blank">Materials and Methods</a> in the presence of increasing concentrations of various co-chaperonin species, as indicated. 100% was taken as the activity of GroEL assisted by a saturating concentration of GroES, or the activity of Cpn60αβ assisted by mt-cpn10. Results are presented as an average of 3 independent experiments ± SD.</p

The oligomeric state of Cpn10(1).

<p>(A) Time-dependent crosslinking pattern of Cpn10(1) (right panel) and Cpn10(2) (left panel). 45 µM of co-chaperonin was exposed to 1 mM DSS for the indicated times. The crosslinking products were separated on a mini gradient SDS acrylamide gels (10–19%), followed by staining with Coomassie blue. The numbers at the right indicate the number of cross-linked subunits in each species. (B) Elution profile of ∼1 mg Cpn10(1) (top panel) or ∼1 mg Cpn10(2) (lower panel) separated by gel filtration on a Superdex 75 column at room temperature at a flow rate of 1 mg/ml. Fractions were analyzed by SDS-PAGE (10 µl per lane). T = 5 µg of Cpn10. Fractions 15–18 contain primarily heptamer while fractions 20–23 contain primarily monomer - dimer.</p

Visualization of Cpn10(1)-Cpn20 hetero-oligomer by native gel.

<p>Co-chaperonin proteins were diluted to 100 µM each (protomer) and then mixed to obtain the indicated ratios. After five minutes at room temperature, native sample buffer was added to a 50 µl final volume and 10 µl of the mixture was loaded on a 6% native acrylamide gel. The identity of the different co-chaperonin species is indicated to the sides of the gel. An additional band above that of Cpn20 can be clearly seen in samples that also contain Cpn10(1). Note that in the native gels, Cpn10(1) appears as three different species.</p

Pf-cpn20 successfully replaces the function of GroES in <i>E. coli</i>.

<p> Complementation assays were carried out in a strain of <i>E. coli</i>, MGM100, in which expression of endogenous chaperonins (GroEL-GroES) was under strict control of the pBAD promoter. (A) Various controls for the <i>in vivo</i> system at the indicated growth conditions (10⁻² dilution shown). (B and C) Ten-fold-serial dilutions (10⁻² to 10⁻⁷ shown) of <i>E. coli</i> strain MGM100 harboring plasmid pOFX containing GroEL and the indicated co-chaperonin, grown on agar plates in the presence of glucose and IPTG, but no arabinose: B) at 30°C C) at 44°C.</p

Pf-cpn20 does not form hetero-oligomers with At-cpn10.

<p>Interaction between Cpn20 and Cpn10 was measured using a pulldown assay. His-tagged At- or Pf-cpn20 was incubated with At-cpn10 and bound to Ni²⁺ beads as described in Materials and Methods. Equivalent aliquots of 12 µl from the total sample (T), unbound fraction (U), fourth wash (W), and bound fraction (B) were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue R-250.</p