title: Interactions of subunit CCT3 in the yeast chaperonin CCT/TRiC with Q/N-rich proteins revealed by high-throughput microscopy analysis.
abstract: 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.
authors.affiliation: Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Nadler-Holly
authors.forename: Michal
authors.initials: M
authors.lastname: Breker
authors.forename: Ranit
authors.initials: R
authors.lastname: Gruber
authors.forename: Ariel
authors.initials: A
authors.lastname: Azia
authors.forename: Melissa
authors.initials: M
authors.lastname: Gymrek
authors.forename: Miriam
authors.initials: M
authors.lastname: Eisenstein
authors.forename: Keith R
authors.initials: KR
authors.lastname: Willison
authors.forename: Maya
authors.initials: M
authors.lastname: Schuldiner
authors.forename: Amnon
authors.initials: A
authors.lastname: Horovitz
mesh_terms: D019943:Amino Acid Substitution; D056404:Chaperonin Containing TCP-1 / Q000235:genetics / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D055550:Protein Stability; D021381:Protein Transport / Q000502:physiology; D020871:RNA Stability / Q000502:physiology; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C512172:CCT3 protein, S cerevisiae; C088425:CCT6 protein, S cerevisiae; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D056404:Chaperonin Containing TCP-1