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. 2006 May 30;103(22):8360-5.
doi: 10.1073/pnas.0600195103. Epub 2006 May 22.

Cytosolic chaperonin protects folding intermediates of Gbeta from aggregation by recognizing hydrophobic beta-strands

Susumu Kubota  1 , Hiroshi Kubota, Kazuhiro Nagata
Affiliations

Cytosolic chaperonin protects folding intermediates of Gbeta from aggregation by recognizing hydrophobic beta-strands

Susumu Kubota et al. Proc Natl Acad Sci U S A. .

Abstract

Cytosolic chaperonin containing t-complex polypeptide 1 (CCT)/TRiC is a group II chaperonin that assists in the folding of newly synthesized proteins. It is a eukaryotic homologue of the bacterial group I chaperonin GroEL. In contrast to the well studied functions of GroEL, the substrate recognition mechanism of CCT/TRiC is poorly understood. Here, we established a system for analyzing CCT/TRiC functions by using a reconstituted protein synthesis by using recombinant elements system and show that CCT/TRiC strongly recognizes WD40 proteins particularly at hydrophobic beta-strands. Using the G protein beta subunit (Gbeta), a WD40 protein that is very rich in beta-sheets, as a model substrate, we found that CCT/TRiC prevents aggregation and assists in folding of Gbeta, whereas GroEL does not. Gbeta has a seven-bladed beta-propeller structure; each blade is formed from a WD40 repeat sequence encoding four beta-strands. Detailed mutational analysis of Gbeta indicated that CCT/TRiC, but not GroEL, preferentially recognizes hydrophobic residues aligned on surfaces of beta-strands in the second WD40 repeat of Gbeta. These findings indicate that one of the CCT/TRiC-specific targets is hydrophobic beta-strands, which are highly prone to aggregation.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
CCT-assisted folding of newly synthesized human β-actin in the PURE system and CCT recognition of Gβ. (A) In vitro translation in the presence of [35S]Met was carried out with or without CCT, GroEL, or GroEL/ES. Radiolabeled products were analyzed by native PAGE with 4.5% gels. (B) After translation of β-actin, DNase I was added. β-Actin–DNase I complex was detected by native PAGE. (C) β-Actin and Gβ translated in the presence of CCT were analyzed by SDS/PAGE (Left) and native PAGE (Right).
Fig. 2.
Fig. 2.
Newly translated Gβ is protected from aggregation by CCT but not by GroEL. (A) CCT prevents Gβ aggregation. Gβ was translated in the presence or absence of CCT or GroEL/GroES. Gβ in the total synthesized fraction (T) and in the supernatant after centrifugation (S) was analyzed by SDS/PAGE, and the percentage in the supernatant fraction was determined (n = 3). (B) CCT protects Gβ from tryptic digestion. Gβ in soluble fractions was digested with l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin for the indicated periods (Upper), and the radioactivity of the Gβ bands was determined (n = 3) (Lower). (C) CCT-assisted folding of Gβ in the presence of Gγ2. Gβ, and Gγ2 were translated in the presence and absence of 35S-Met, respectively. These samples were mixed and then subjected to tryptic digestion. The full-length and trypsin-resistant portions of Gβ are indicated by the arrowhead and arrows, respectively. Asterisks indicate significant differences (see Materials and Methods).
Fig. 3.
Fig. 3.
The WD2 repeat of Gβ is strongly and specifically recognized by CCT but not by GroEL. (A) Schematic representation of human Gβ1 structure. This model is based on the β-propeller-containing crystal structure of bovine Gβ1. Each of the seven WD40 repeats (WD1–WD7) contains four-stranded β-sheets. (B) Sequence alignment of the seven WD40 repeats of the human Gβ1. Amino acid residues constituting β-strand structures are underlined. Negatively and positively charged residues are shown in red and blue, respectively. (C) CCT binding to Gβ deletion mutants. Gβ deletion mutants were translated in the presence of CCT, and soluble fractions were analyzed by SDS/PAGE and native PAGE. (D) CCT binding to individual WD40 repeats of Gβ (n = 3). (E) GroEL binding to individual WD40 repeats of Gβ. NS, nonspecific bands. Arrows indicate CCT-bound or GroEL-bound products. ND, not determined.
Fig. 4.
Fig. 4.
The β3 region of WD2 is strongly recognized by CCT. (A) The deletion of β3 diminishes the binding of WD2 to CCT. WD2 and its deletion mutants shown in Fig. 12A were translated in the presence of CCT, and the soluble fractions were subjected to tricine-SDS/PAGE and native PAGE. CCT-binding ratios are shown at the bottom (n = 3). (B) The β3 region of WD2 significantly enhances CCT binding affinity of other WD40 repeats. The β3 region of WD3 or WD5 was replaced by the β3 of WD2, as shown in Fig. 12B, and CCT-binding ratios were determined (n = 3). NS, nonspecific bands. ND, not determined.
Fig. 5.
Fig. 5.
CCT recognizes the φ-x-φ-x-φ-x sequences in the β3 region of WD40 repeats, where φ is a hydrophobic residue. (A) Single amino acids in the β3 region of WD2-Δβ4 were substituted with lysine, and these mutants were analyzed for CCT binding (n = 3). (B and C) The S122 residue of WD2-Δβ4 was substituted with negatively charged (B) or hydrophobic (C) residues (n = 3). (D) Converting WD3, WD5, or WD6 to φ-x-φ-x-φ-x sequence-containing WD40 repeats significantly enhances their binding to CCT (n = 3).
Fig. 6.
Fig. 6.
CCT recognizes the β1 of WD2 through hydrophobic interactions. The indicated single (A and B) or double (C) mutations were introduced into WD2 and analyzed for CCT binding (n = 3).
Fig. 7.
Fig. 7.
WD2 secondary structure is likely to be required for CCT recognition. (A) Single amino acid substitutions were introduced into the loop bridging the β2 and β3 regions of WD2, and these mutants were analyzed for CCT binding (n = 3). (B) The position of the D118 residue that stabilizes the β-sheet structure of WD2. (C) Location of the residues that form the β3 of WD2 in the molecular structure of Gβ. The φ and x residues of the φ-x-φ-x-φ-x (I120 to N125) sequence in the β3 region of WD2 are indicated by the red and blue space-filled models, respectively.
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