Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Interviews
    • Sign up for alerts
  • About us
    • About BiO
    • Editors and Board
    • Editor biographies
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contact
    • Contact BiO
    • Advertising
    • Feedback
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

User menu

  • Log in

Search

  • Advanced search
Biology Open
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

supporting biologistsinspiring biology

Biology Open

Advanced search

RSS   Twitter   Facebook   YouTube

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Interviews
    • Sign up for alerts
  • About us
    • About BiO
    • Editors and Board
    • Editor biographies
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contact
    • Contact BiO
    • Advertising
    • Feedback
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
Jorge Cuellar
1Centro Nacional de Biotechnología (CNB-CSIC), Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hugo Yébenes
1Centro Nacional de Biotechnología (CNB-CSIC), Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain
*Present address: Centro de Investigaciones Biológicas (CIB-CSIC), 28040 Madrid, Spain.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sandra K. Parker
2Department of Marine and Environmental Sciences and Department of Biology, Northeastern University, Marine Science Center, Nahant, MA 01908, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gerardo Carranza
3Departamento de Biología Molecular, Universidad de Cantabria-IFIMAV, 39011 Santander, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Marina Serna
1Centro Nacional de Biotechnología (CNB-CSIC), Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
José María Valpuesta
1Centro Nacional de Biotechnología (CNB-CSIC), Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Juan Carlos Zabala
3Departamento de Biología Molecular, Universidad de Cantabria-IFIMAV, 39011 Santander, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
H. William Detrich III
2Department of Marine and Environmental Sciences and Department of Biology, Northeastern University, Marine Science Center, Nahant, MA 01908, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: iceman@neu.edu
  • Article
  • Figures & tables
  • Info & metrics
  • eLetters
  • PDF
Loading

Article Figures & Tables

Figures

  • Fig. 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    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.
    • Download figure
    • Open in new tab
    • Download powerpoint
    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.
    • Download figure
    • Open in new tab
    • Download powerpoint
    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.
    • Download figure
    • Open in new tab
    • Download powerpoint
    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.
    • Download figure
    • Open in new tab
    • Download powerpoint
    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.
    • Download figure
    • Open in new tab
    • Download powerpoint
    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.
Previous ArticleNext Article
Back to top
Previous ArticleNext Article

This Issue

RSSRSS

Keywords

  • CCT
  • TriC
  • Chaperone
  • Chaperonin
  • Protein folding
  • Actin
  • Tubulin
  • Thermal adaptation
  • Evolution

 Download PDF

Email

Thank you for your interest in spreading the word on Biology Open.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Assisted protein folding at low temperature: evolutionary adaptation of the Antarctic fish chaperonin CCT and its client proteins
(Your Name) has sent you a message from Biology Open
(Your Name) thought you would like to see the Biology Open web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
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
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
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

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Alerts

Please log in to add an alert for this article.

Sign in to email alerts with your email address

Article Navigation

  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • RESULTS
    • DISCUSSION
    • MATERIALS AND METHODS
    • Acknowledgements
    • Funding
    • Footnotes
    • References
  • Figures & tables
  • Info & metrics
  • eLetters
  • PDF

Related articles

Cited by...

More in this TOC section

  • Wound-induced polyploidization is dependent on Integrin-Yki signaling
  • A stem cell based in vitro model of NAFLD enables the analysis of patient specific individual metabolic adaptations in response to a high fat diet and AdipoRon interference
  • Mutations in the splicing regulator Prp31 lead to retinal degeneration in Drosophila
Show more RESEARCH ARTICLE

Similar articles

Other journals from The Company of Biologists

Development

Journal of Cell Science

Journal of Experimental Biology

Disease Models & Mechanisms

Advertisement

Biology Open and COVID-19

We are aware that the COVID-19 pandemic is having an unprecedented impact on researchers worldwide. The Editors of all The Company of Biologists’ journals have been considering ways in which we can alleviate concerns that members of our community may have around publishing activities during this time. Read about the actions we are taking at this time.

Please don’t hesitate to contact the Editorial Office if you have any questions or concerns.


2020 at The Company of Biologists

Despite 2020’s challenges, we achieved a lot at The Company of Biologists. In the midst of the pandemic, we have seen long-term projects and new ventures come to fruition. Read our full lowdown of 2020.


Interview- Sebastian Markert

Sebastian Markert is first author of a paper in BiO using C. elegans to model amyotrophic lateral sclerosis. In an interview, he talks about the potential implications of his work and his future plans.


Three communities to support biologists to everywhere

Online communities have never been more important. If you’re looking for somewhere to meet fellow scientists, take part in topical discussions and find virtual events in your field, take a look at each of our community sites:

  • The Node: the community site for and by developmental biologists
  • preLights: the preprint highlights service run by the biological community
  • FocalPlane: the community site for microscopists and biologists alike

Articles

  • Accepted manuscripts
  • Issue in progress
  • Latest complete issue
  • Issue archive
  • Archive by article type
  • Interviews
  • Sign up for alerts

About us

  • About BiO
  • Editors and Board
  • Editor biographies
  • Grants and funding
  • Journal Meetings
  • Workshops
  • The Company of Biologists

For Authors

  • Submit a manuscript
  • Aims and scope
  • Presubmission enquiries
  • Article types
  • Manuscript preparation
  • Cover suggestions
  • Editorial process
  • Promoting your paper
  • Open Access

Journal Info

  • Journal policies
  • Rights and permissions
  • Media policies
  • Reviewer guide
  • Sign up for alerts

Contact

  • Contact BiO
  • Advertising
  • Feedback

Twitter   YouTube   LinkedIn

© 2021   The Company of Biologists Ltd   Registered Charity 277992