Interactions of subunit CCT3 in the yeast chaperonin CCT/TRiC with Q/N-rich proteins revealed by high-throughput microscopy analysis

Proc Natl Acad Sci U S A. 2012 Nov 13;109(46):18833-8. doi: 10.1073/pnas.1209277109. Epub 2012 Oct 29.


The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is an ATP-fueled machine that assists protein folding. It consists of two back-to-back stacked rings formed by eight different subunits that are arranged in a fixed permutation. The different subunits of CCT are believed to possess unique substrate binding specificities that are still mostly unknown. Here, we used high-throughput microscopy analysis of yeast cells to determine changes in protein levels and localization as a result of a Glu to Asp mutation in the ATP binding site of subunits 3 (CCT3) or 6 (CCT6). The mutation in subunit CCT3 was found to induce cytoplasmic foci termed P-bodies where mRNAs, which are not translated, accumulate and can be degraded. Analysis of the changes in protein levels and structural modeling indicate that P-body formation in cells with the mutation in CCT3 is linked to the specific interaction of this subunit with Gln/Asn-rich segments that are enriched in many P-body proteins. An in vitro gel-shift analysis was used to show that the mutation in subunit CCT3 interferes with the ability of CCT to bind a Gln/Asn-rich protein aggregate. More generally, the strategy used in this work can be used to unravel the substrate specificities of other chaperone systems.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Chaperonin Containing TCP-1 / genetics
  • Chaperonin Containing TCP-1 / metabolism*
  • Cytoplasmic Granules / genetics
  • Cytoplasmic Granules / metabolism
  • Mutation, Missense
  • Protein Stability
  • Protein Transport / physiology
  • RNA Stability / physiology
  • RNA, Fungal / genetics
  • RNA, Fungal / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*


  • CCT3 protein, S cerevisiae
  • CCT6 protein, S cerevisiae
  • RNA, Fungal
  • RNA, Messenger
  • Saccharomyces cerevisiae Proteins
  • Chaperonin Containing TCP-1