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Research Article
Assisted protein folding at low temperature: evolutionary adaptation of the Antarctic fish chaperonin CCT and its client proteins
Jorge Cuellar, Hugo Yébenes, Sandra K. Parker, Gerardo Carranza, Marina Serna, José María Valpuesta, Juan Carlos Zabala, H. William Detrich III
Biology Open 2014 3: 261-270; doi: 10.1242/bio.20147427

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Figures

  • Fig. 1.
    Fig. 1. Purification of G. gibberifrons CCT from immature testis tissue and separation of subunits by HPLC.

    An ammonium sulfate cut (30–50%) of a centrifugal extract of testis tissue was chromatographed on Heparin Sepharose, CCT-containing fractions were pooled and centrifuged on sucrose gradients, and the ∼25S chaperonin fraction was chromatographed on Superose 6 (see Materials and Methods for details). The subunits of CCT were isolated by HPLC for subsequent analysis by mass spectrometry (Fig. 2). Throughout the purification, protein compositions of fractions were analyzed by SDS-PAGE. (A) Testis extract, dominated by α- and β-tubulins, prior to application to Heparin Sepharose. (B) Fractions containing CCT (subunits ∼55–60 kDa) eluted from the Heparin Sepharose column by a 0.45→0.6 M NaCl step gradient. (C,D) CCT-enriched fractions from Heparin chromatography (B) were pooled and then centrifuged through 10–50% sucrose gradients (C), and proteins sedimenting at ∼25 S were analyzed by electrophoresis (D). (E) Pooled CCT was loaded on a Superose 6 gel filtration column, and the material eluting at an Mr of ∼106, which consisted of nearly homogeneous CCT, was collected. (F) HPLC elution profile of CCT subunits and several contaminating proteins; the starting material corresponded to purified CCT shown in Fig. 1D. (G) SDS-PAGE of HPLC fractions on 8.5% gels. Protein identities were established by mass spectrometry (Fig. 2): α–θ, CCT subunits; EF1α, elongation factor 1α; SHMT, serine hydroxymethyltransferase; α-tub, β-tub, α- and β-tubulins, respectively. Absorbance (mAU) and the solvent gradient (%B) are plotted vs elution volume in panel F. Note that CCT subunits γ and ε were each resolved as two peaks.

  • Fig. 2.
    Fig. 2. Identification of G. gibberifrons CCT subunits.

    Plugs containing the indicated protein bands were excised from the gel shown in Fig. 1G for in-gel tryptic proteolysis and mass spectrometric analysis. The identities of the presumptive G. gibberifrons CCT subunits were confirmed by querying the non-redundant NCBI protein database with the G. gibberifrons tryptic peptide sets. Each subunit possessed six, seven, or eight peptides that mapped perfectly to peptides of a bovine CCT subunit (red). Peptide sequence coverage ranged from 15–27% for the G. gibberifrons/bovine comparison, and higher values were found for comparison to CCT subunits from other fishes (data not shown). Percent coverage was highest for the β (67%) and θ (55%) subunits of CCT from the Antarctic Bullhead notothen, N. coriiceps, whose sequences had been established previously from cloned cDNAs (Pucciarelli et al., 2006). The calculated pIs correspond to the bovine CCT subunits.

  • Table 1. Isoelectric points of CCT subunits from an Antarctic fish and a mammal
    Table 1.
  • Fig. 3.
    Fig. 3. Structural characterization of G. gibberifrons CCT by EM: comparison to the bovine chaperonin.

    Two-dimensional average images of apo- and holo-CCTs were generated as described in Materials and Methods. (A) apo-CCT from G. gibberifrons, top view (n = 575 particles). (B) apo-CCT from G. gibberifrons, side view (n = 486 particles). (C) G. gibberifrons CCT in complex with N. coriiceps β1-tubulin, top view (n = 650 particles). (D) CCT–tubulin complex from the cow, top view (n = 570 particles). (E) G. gibberifrons CCT in complex with C. aceratus actin, top view (n = 710 particles). (F) CCT–actin complex from the cow, top view (n = 657 particles). Scale bar: 5 nm.

  • Fig. 4.
    Fig. 4. G. gibberifrons CCT binds to, folds, and releases C. aceratus actin at physiological temperature.

    CCT was incubated with denatured actin at intervals from 0 to 96 h at 2°C in binding buffer containing 1 mM Mg2+-ATP. Reaction products were analyzed at 2°C by non-denaturing electrophoresis on 4.5% polyacrylamide gels followed by autoradiography. (A) apo-CCT migrates as a single band as shown on this Coomassie Blue-stained gel. (B–D) Folded β-actin is detected at 12 h and increases in amount until a plateau is reached at 72–96 h. Large amounts of β-actin remained in complex with CCT. The positions of apo-CCT, of CCT–β-actin, and of folded actin monomer are indicated.

  • Fig. 5.
    Fig. 5. Temperature dependence of CP binding by testis CCTs from an Antarctic notothen and the cow.

    The binding of CPs by their homologous chaperonins was examined at four temperatures between −4°C and +20°C. Experiments were performed in triplicate, and at least 2000 top-view CCT particles from each binding reaction were scored automatically as apo- or holo-CCT as described in Materials and Methods. Data are presented as percentage CCT bound to CP (mean ± s.d.). In toto, >110,000 particles were scored. Client proteins: (A) actin; (B) tubulin. Chaperonins: hatched bars, G. gibberifrons; black bars, Bos taurus (cow).

  • Fig. 6.
    Fig. 6. Temperature dependence of CP binding by CCT in homologous and heterologous combinations.

    CCT–CP binding reactions were performed and analyzed as described in Materials and Methods. (A,B) Homologous combinations of CCT and CP: actin (A); tubulin (B). (C,D) Heterologous combinations of CCT and CP: actin (C); tubulin (D). Incubation temperatures are given beneath each bar. Red bars, incubations performed at 20°C; blue bars, incubations performed at 4°C. Data are presented as percentage CCT bound to CP. Abbreviations: Act, actin; AF, Antarctic fish; Bt, B. taurus; Gg, G. gibberifrons (notothen); Tub, tubulin.

  • Table 2. Temperature dependence of the ATPase activities of apoCCTs from an Antarctic fish and a mammal
    Table 2.
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Research Article
Assisted protein folding at low temperature: evolutionary adaptation of the Antarctic fish chaperonin CCT and its client proteins
Jorge Cuellar, Hugo Yébenes, Sandra K. Parker, Gerardo Carranza, Marina Serna, José María Valpuesta, Juan Carlos Zabala, H. William Detrich III
Biology Open 2014 3: 261-270; doi: 10.1242/bio.20147427
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