Equivalent Mutations in the Eight Subunits of the Chaperonin CCT Produce Dramatically Different Cellular and Gene Expression Phenotypes

Amit, Maya; Weisberg, Sarah J.; Nadler-Holly, Michal; McCormack, Elizabeth A.; Feldmesser, Ester; Kaganovich, Daniel; Willison, Keith R.; Horovitz, Amnon

doi:10.1016/j.jmb.2010.06.037

Cited by 91 publications

(87 citation statements)

References 41 publications

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“…The S. cerevisiae (S288C) Y6545 strains with the mutations D91E and D89E in subunits CCT3 and CCT6, respectively, were generated by homologous recombination as previously described (14). These two query strains with mutations in subunits CCT3 or CCT6 were crossed using the synthetic genetic array (SGA) methodology (15,19) with a complete library of haploid strains expressing endogenous yeast proteins fused at their C termini to GFP (17), thereby yielding two libraries designated throughout this article as MA3 and MA6, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…Light gray, dark gray, and black designate Q, N, and all other residues, respectively. described (14), and the resulting strains are designated here as MA3 and MA6. A library of haploid S. cerevisiae strains, each expressing an endogenous locus yeast protein tagged in-frame at its C terminus to GFP (17), was crossed with these MA3 and MA6 query strains.…”

Section: Methodsmentioning

confidence: 99%

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

Nadler-Holly¹,

Breker²,

Gruber³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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 highthroughput 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. molecular chaperones | polyQ proteins | protein mis-folding | protein aggregation | high-content analysis C haperonins are ATP-dependent protein-folding machines that are present in all kingdoms of life. They consist of two back-toback stacked oligomeric rings with a cavity at each end, where protein substrate binding and folding take place (for reviews see refs. 1 and 2). The chaperonins can be divided into two groups: group I, found in the bacterial cytoplasm (e.g., GroEL in Escherichia coli), mitochondria, and chloroplasts; and group II found in archaea and the eukaryotic cytosol. Numerous studies have shown that the group I chaperonin GroEL can facilitate the folding of a large number of different proteins in vitro, although its role in the cell is much more limited (for review see ref.3). The group II eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/ TRiC) also seems to have a specialized role in vivo in the folding of actin (4), tubulin (5), and other essential proteins, including regulators of cell division and cytoskeleton formation (6-8), despite seeming to possess broad binding specificity (6). The list of members in the CCT interactome is, however, not yet fully established, and the conditions for entry into this exclusive club remain poorly understood.An important distinction between group I and group II chaperonins is that the former consist of homo-oligomeric rings, whereas the latter usually consist of hetero-oligomeric rings that contain two or three different subunits in the case of many archaeal chaperonins and eight different subunits in the case of CCT. The eight subunits of CCT are arranged in a defined permutation (9), and the orientation of the two rings of CCT with respect to each other is also fixed (10). CCT's heter...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Nadler-Holly¹,

Breker²,

Gruber³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…The subunit specialization occurred very early in eukaryote evolution (11) and is conserved to such an extent that the sequence identity between mammalian and yeast subunits of the same type is nearly 60%, whereas the sequence identity between the different subunit types in the same organism is only 30%. The diversity of eight distinct subunits allows for specific substrate binding modes (4, 12, 13) as well as other cycle-related activities such as differential ATP hydrolysis (14,15).…”

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confidence: 99%

Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

Kalisman

Adams

Levitt

2012

Proc. Natl. Acad. Sci. U.S.A.

164

178

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The TRiC/CCTchaperonin is a 1-MDa hetero-oligomer of 16 subunits that assists the folding of proteins in eukaryotes. Low-resolution structural studies confirmed the TRiC particle to be composed of two stacked octameric rings enclosing a folding cavity. The exact arrangement of the different proteins in the rings underlies the functionality of TRiC and is likely to be conserved across all eukaryotes. Yet despite its importance it has not been determined conclusively, mainly because the different subunits appear nearly identical under low resolution. This work successfully addresses the arrangement problem by the emerging technique of cross-linking, mass spectrometry, and modeling. We cross-linked TRiC under native conditions with a cross-linker that is primarily reactive toward exposed lysine side chains that are spatially close in the context of the particle. Following digestion and mass spectrometry we were able to identify over 60 lysine pairs that underwent crosslinking, thus providing distance restraints between specific residues in the complex. Independently of the cross-link set, we constructed 40,320 (¼8 factorial) computational models of the TRiC particle, which exhaustively enumerate all the possible arrangements of the different subunits. When we assessed the compatibility of each model with the cross-link set, we discovered that one specific model is significantly more compatible than any other model. Furthermore, bootstrapping analysis confirmed that this model is 10 times more likely to result from this cross-link set than the next best-fitting model. Our subunit arrangement is very different than any of the previously reported models and changes the context of existing and future findings on TRiC.group II chaperonins | protein folding | violation distances I n eukaryotes and archaea the folding of nascent and mis-folded polypeptide chains is assisted by a group II chaperonin system known as the thermosome in archea and TRiC (or CCT) in eukaryotes (1). These large protein complexes consist of two stacked octameric rings (2) The open state of the complex binds the polypeptide substrate (4, 5), whereupon closing the substrate is sequestered into a large interior cavity where folding can occur. TRiC has been implicated in the folding pathways of many cytosolic proteins (6), most notably actin and tubulin (7,8).Many archaeal species have just one thermosome gene and a simple homo-oligomeric architecture (9). Other archaeal species have at most three different types of subunits, and there is evidence that the multiple genes are redundant (10). In stark contrast, the eukaryotic TRiC consists of eight different subunit types (CCT1 to CCT8), all of which are essential. The subunit specialization occurred very early in eukaryote evolution (11) and is conserved to such an extent that the sequence identity between mammalian and yeast subunits of the same type is nearly 60%, whereas the sequence identity between the different subunit types in the same organism is only 30%. The diversity of eight distinct subun...

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“…6). It is clear, however, that there is a core face formed by the contiguous subunits CCT5/CCT2/CCT4, in which the phenotypic effect of mutations in the ATP-binding site is severe [103]; these subunits have high ATP binding and hydrolysis activity. Another core face apposed to these subunits is formed by subunits CCT8/CCT6/CCT3, with no notable phenotypic effect caused by mutations in their ATP-binding site and which have low or negligible ATP binding and hydrolysis activities.…”

Section: Conformational Changes and Reaction Cyclementioning

confidence: 99%

Dynamics, flexibility, and allostery in molecular chaperonins

et al. 2015

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a b s t r a c tThe chaperonins are a family of molecular chaperones present in all three kingdoms of life. They are classified into Group I and Group II. Group I consists of the bacterial variants (GroEL) and the eukaryotic ones from mitochondria and chloroplasts (Hsp60), while Group II consists of the archaeal (thermosomes) and eukaryotic cytosolic variants (CCT or TRiC). Both groups assemble into a dual ring structure, with each ring providing a protective folding chamber for nascent and denatured proteins. Their functional cycle is powered by ATP binding and hydrolysis, which drives a series of structural rearrangements that enable encapsulation and subsequent release of the substrate protein.Chaperonins have elaborate allosteric mechanisms to regulate their functional cycle. Long-range negative cooperativity between the two rings ensures alternation of the folding chambers. Positive intra-ring cooperativity, which facilitates concerted conformational transitions within the protein subunits of one ring, has only been demonstrated for Group I chaperonins. In this review, we describe our present understanding of the underlying mechanisms and the structurefunction relationships in these complex protein systems with a particular focus on the structural dynamics, allostery, and associated conformational rearrangements.

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Equivalent Mutations in the Eight Subunits of the Chaperonin CCT Produce Dramatically Different Cellular and Gene Expression Phenotypes

Cited by 91 publications

References 41 publications

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

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

Subunit order of eukaryotic TRiC/CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling

Dynamics, flexibility, and allostery in molecular chaperonins

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