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Review
. 2018 Jun 19;373(1749):20170192.
doi: 10.1098/rstb.2017.0192.

The substrate specificity of eukaryotic cytosolic chaperonin CCT

Keith R Willison  1
Affiliations
Review

The substrate specificity of eukaryotic cytosolic chaperonin CCT

Keith R Willison. Philos Trans R Soc Lond B Biol Sci. .

Abstract

The cytosolic chaperonin CCT (chaperonin containing TCP-1) is an ATP-dependent double-ring protein machine mediating the folding of members of the eukaryotic cytoskeletal protein families. The actins and tubulins are obligate substrates of CCT because they are completely dependent on CCT activity to reach their native states. Genetic and proteomic analysis of the CCT interactome in the yeast Saccharomyces cerevisiae revealed a CCT network of approximately 300 genes and proteins involved in many fundamental biological processes. We classified network members into sets such as substrates, CCT cofactors and CCT-mediated assembly processes. Many members of the 7-bladed propeller family of proteins are commonly found tightly bound to CCT isolated from human and plant cells and yeasts. The anaphase promoting complex (APC/C) cofactor propellers, Cdh1p and Cdc20p, are also obligate substrates since they both require CCT for folding and functional activation. In vitro translation analysis in prokaryotic and eukaryotic cell extracts of a set of yeast propellers demonstrates their highly differential interactions with CCT and GroEL (another chaperonin). Individual propeller proteins have idiosyncratic interaction modes with CCT because they emerged independently with neo-functions many times throughout eukaryotic evolution. We present a toy model in which cytoskeletal protein biogenesis and folding flux through CCT couples cell growth and size control to time dependent cell cycle mechanisms.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

Keywords: 7-bladed WD40 propellers; APC/C; actin; chaperonin CCT; protein folding; tubulin.

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Conflict of interest statement

We declare we have no competing interests.

Figures

Figure 1.
Figure 1.
Cartoons of schemes for CCT action upon its substrates and binding complex formation. (a) Direct folding to native state: actin filaments (F-actin) are composed of a single monomer species (G-actin) and CCT activity yields assembly competent monomers. It is not yet known if ATP loading is concomitant with folding or whether nucleotide-free actin is released into the 2–5 mM ATP pool present in cell cytoplasm and then equilibrates with free ATP. (b) Folding and assembly: tubulin filaments are composed of two monomer species (α-tubulin and β-tubulin) which both bind guanosine triphosphate (GTP). GTP concentration is low in cells (50 µM) and CCT may well play a direct role in GTP loading of β-tubulin. There is strong evidence for post-CCT-dependent folding and assembly step(s), involving several cofactor proteins, which produce filament-assembly competent tubulin monomers. (c) Production of the APC activators: Cdc20p and Cdh1p propeller proteins are CCT-dependent. (d) Assembly of G-protein complexes [18]. (e) Type II phosphatase complex—CCT interaction conserved from yeast to humans. (f) TORC complex: WD40 subunits. (g) Secretory complexes ER and Golgi. (h) Transcription factor and several histone deacetylase complexes interact with CCT [19]. (i) RNA processing. (j) Holding activity of the Saccharomyces cerevisiae Vid27p propeller protein involved in autophagy. (Online version in colour.)
Figure 2.
Figure 2.
The Velcro cap. Structural model of the propeller of TUP1 (PDB: 1erj). TUP1 is a 713-amino acid residue protein with a single 7-bladed propeller. The left-hand panel shows a ribbon diagram with the W, D and H residues of the WD40 repeat highlighted in pink which interact to form a structural element connecting strands 7A and 7C across which strand 7D binds like a ‘Velcro’ cap [33]. The two right-hand panels show top and side views of the propeller, a conical frustum. The amino acid sequences of the interaction region between the β-sheets are shown below, with the four sequences aligned to reflect the registration of the blades in three-dimensional space (KR Willison 2011, unpublished analysis).
Figure 3.
Figure 3.
Screening WD40-repeat proteins for CCT interaction. (a) 6% native PAGE analysis of TAF5, UTP13, ELP2 and DOA1 after in vitro translation in E. coli lysate (E) or rabbit reticulocyte lysate (R) in vitro transcription/translation system to detect GroEL (filled arrowhead) or CCT interaction (open arrowhead) respectively. (b) Graphs of time courses of CCT-bound protein by 6% native PAGE analysis of rabbit reticulocyte in vitro translation assays of TAF5, UTP13 and HAT2. (c) 6% native PAGE analysis of time course of rabbit reticulocyte in vitro translation assay of VID27. Inset image of sample at the end of the time course showing interaction of CCT with Vid27p which can be shifted with the anti-TCP1 antibody 23C, demonstrating specificity of CCT binding. Vid27p is the most avid CCT binding protein in yeast [19,25,46]. Vid27 is a little studied, non-essential, WD40-repeat protein most closely related to BUB3 and possibly involved in vacuolar protein degradation and is found in yeasts and plants but not in higher eukaryotes.
Figure 4.
Figure 4.
Mapping CCT-binding residues in the Cdh1p propeller. Mutants in the Cdh1p propeller (amino acid residues 226–566) were screened for strength of binding and degree of processing by CCT using in vitro translation/native gel analysis. Six mutations were tested in the context of the propeller domain only. (a) The β-strands in the blades are highlighted in green. Motif: the red bold letters highlight the residues (GH-G-D-WD) forming the hydrogen-bonding network (‘structural tetrad’) which stabilizes the blades. The M332A mutation, coloured yellow, is included to prevent internal translation initiation at this methionine which reduces the accuracy of quantitation in these assays [27]; the equivalent residue in human CDH1 is alanine. The point mutants which arrest the processing are highlighted in magenta. (b) List of 14 mutants and their CCT interaction behaviour. Strong arrest indicates that greater than 15% of the counts become associated with CCT; slow processing means that the appearance of the folded Cdh1 (either full-length or the propeller domain) is retarded compared to wild-type control.
Figure 5.
Figure 5.
Yeast CCT capacity. Number estimates for the components of this yeast model are from the following sources. (a) Calculated mRNA copy numbers per cell based upon 15 000 transcripts per cell growing at 30°C - ACT1: 28.97, CDH1: 0.36, TUB2: 5.94, TCP1: 1.81, CCT2: 1.56, CCT3: 1.62, CCT4: 2.16, CCT5: 2.24, CCT6: 2.61, CCT7: 2.23, CCT8: 1.7, CDH1: 0.36 [30]. Red squiggles represent estimate of steady state number of nascent chains bound to CCT. (b) Haploid versus diploid protein ratios for CCT subunits (1–8), prefoldin subunits (9–14), ACT1 (15), TUB2 (16), PLP2 (17) taken from de Godoy et al. [31]. (c,d) Half-live distributions (hours) and steady-state abundance distributions (arbitrary units) of 641 proteins [53] are plotted according to increasing value on the x-axis. Half-lives and adundances of CCT subunits (red dots), ACT1 (yellow dot) and TUB2 (blue dot) are highlighted on each distribution. (Online version in colour.)
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References

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