1. Functional Partition of Cpn60α and Cpn60β Subunits in Substrate Recognition and Cooperation with Co-chaperonins 
Shijia Zhang, Huan Zhou, Feng Yu, Feng Gao, Jianhua He, Cuimin Liu 
Molecular Plant 
Volume 9, Issue 8, Pages (August 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
Functional Divergence of Plastidic CPN60 Subunits Determined by Their Apical Domains.
(A) (Left) Diagram of chaperonin subunits and the constructed chaperonin chimeras. Domain designation and amino acid numbering were used with respect to GroEL. E1 domain (1–137), I1 domain (138–190), A (191–374), I2 (375–409), E2 (410–548). (Right) Functional complementation of GroEL by chaperonin chimeras. The GroEL/ES-deficient E. coli strain MGM100 was transformed with individual chaperonin chimeras and GroES, and grown on medium supplemented with glucose and IPTG at 37°C for 13 h.
(B) Analytical gel filtration of chaperonin chimeras and GroES complexes. 50 μl of reaction mixture containing 2 μM chaperonin chimeras and 10 μM GroES was run on a Superdex 200 column (PC3.2/10) with buffer containing 50 μM ADP-AlF3. Collected fractions were resolved by SDS–PAGE and stained with Coomassie.
(C) The binding efficiency of denatured RbcL to chaperonin chimeras. 50 μM chemically denatured Rubisco from Rhodospirillum rubrum (RrRubisco) or Chlamydomonas reinhardtii (CrRubisco) was diluted 100-fold into buffer with or without 1 μM chaperonin chimeras present. Rubisco in the supernatant was analyzed by SDS–PAGE and immunoblotting. Native Rubisco was loaded as control. The relative signal density was quantified with Quantity One (Bio-Rad) with the binding efficiency of GroEL set to 100%. The binding efficiency is indicated below each lane as calculated from three independent experiments.
(D) The crystal structures of apical domains from CPN60α and CPN60β1. The structure of CPN60α is colored cyan and the structure of CPN60β1 is colored pink. The helices H and I are enlarged. The distance between atoms of the first Cα of the two helices is shown in CPN60α and CPN60β1 in cyan and pink, respectively.
(E) Functional complementation by chimera A1 point mutants: Q203Y, T241I, and Q203Y/T241I. The experiments were performed as described for (A).
(F) The binding efficiency of denatured CrRbcL to chaperonin chimera A1 or its point mutants (Q203Y, T241I, Q203Y/T241I). The experiments were performed as described for (C) except that the binding efficiency is shown in the graph below as calculated from three independent experiments, with error bars indicating SD.
(G) (Left) Diagram of Cpn60 subunits and the constructed Cpn60 chimeras. The domain designation and amino acid numbering are as described in (A). (Right) GroEL functional complementation by co-expression of Cpn60 subunits and its chimeras, as well as its plastidic co-chaperonin CPNs (consisting of CPN23, CPN20, and CPN11).
(H) ATPase activity of CPN60 oligomers. The ATP hydrolysis rate of 0.2 μM different CPN60 oligomers in the presence or absence of 0.4 μM GroES or CPNs was measured for 10 min at 25°C.
(I) The binding efficiency of denatured CrRbcL to CPN60 oligomers. The experiments were performed as described for (F).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions