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. 1997 May 13;94(10):5383-8.
doi: 10.1073/pnas.94.10.5383.

Chaperonin filaments: the archaeal cytoskeleton?

J D Trent  1 , H K Kagawa, T Yaoi, E Olle, N J Zaluzec
Affiliations

Chaperonin filaments: the archaeal cytoskeleton?

J D Trent et al. Proc Natl Acad Sci U S A. .

Abstract

Chaperonins are high molecular mass double-ring structures composed of 60-kDa protein subunits. In the hyperthermophilic archaeon Sulfolobus shibatae the two chaperonin proteins represent approximately 4% of its total protein and have a combined intracellular concentration of >30 mg/ml. At concentrations >/= 0.5 mg/ml purified chaperonins form filaments in the presence of Mg2+ and nucleotides. Filament formation requires nucleotide binding (not hydrolysis), and occurs at physiological temperatures in biologically relevant buffers, including a buffer made from cell extracts. These observations suggest that chaperonin filaments may exist in vivo and the estimated 4600 chaperonins per cell suggest that such filaments could form an extensive cytostructure. We observed filamentous structures in unfixed, uranyl-acetate-stained S. shibatae cells, which resemble the chaperonin filaments in size and appearance. ImmunoGold (Janssen) labeling using chaperonin antibodies indicated that many chaperonins are associated with insoluble cellular structures and these structures appear to be filamentous in some areas, although they could not be uranyl-acetate-stained. The existence of chaperonin filaments in vivo suggests a mechanism whereby their protein-folding activities can be regulated. More generally, the filaments themselves may play a cytoskeletal role in Archaea.

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Figures

Figure 1
Figure 1
Above a critical concentration freshly purified S. shibatae chaperonins form filaments at room temperature in the presence of Mg2+. (A) Purified chaperonin proteins in a HKM buffer appear as double rings at concentrations of 0.1 mg/ml; (B) rings and short chains are seen at 0.5 mg/ml; and (C) long chains and filaments are present at 1.0 mg/ml. At chaperonin concentrations of 1.0 mg/ml filaments did not form in Hepes buffer alone (D), or when 10 mM KCl was added (E), but did form when 10 mM MgCl2 was added (F).
Figure 2
Figure 2
Chaperonins stored for >48 h require Mg2+ and nucleotides to form filaments at a physiological temperature. (A) “Aged” chaperonin (1 mg/ml) in Hepes buffer (pH 7.5) and 25 mM MgCl2 after 1 h at 75°C; (B) the same chaperonin sample with 1 mM ATP added; (C) with 1 mM ADP added; and (D) with 1 mM AMP-PNP added.
Figure 3
Figure 3
Chaperonin filament formation at 75°C in the presence of Mg2+ and nucleotides in different buffers measured by light scattering. (A) Changes in absorption (350 nm) of purified chaperonin (1.0 mg/ml) in Hepes buffer after the addition (inject) of 25 mM MgCl2 and 1.0 mM ATP, ADP, or AMP-PNP; (B) changes in absorption of purified chaperonin in a complex buffer made from cell extract (see Results) after the addition of MgCl2 and ATP (inject); the cell extract buffer itself (control) increased in absorption due to aggregation, which was suppressed by the addition of chaperonins but not after the chaperonins formed filaments. TEM analysis of samples before and after the addition of nucleotides indicate that the increased absorption in chaperonin samples is correlated with the presence of filaments.
Figure 4
Figure 4
Micrographs of intracellular filaments in detergent-treated, unfixed S. shibatae cells lightly stained with uranyl acetate, taken using intermediate voltage TEM (100–300 kV). A low (A) and high (A′) magnification of the same cell shows the distribution and distinct periodic structure of the intracellular filaments (arrowheads). Similar filaments were seen in many cells and four representative micrographs are shown (B–E).
Figure 5
Figure 5
ImmunoGold labeling of detergent-treated S. shibatae cells with polyclonal antibodies against the chaperonin seen at low (A) and high (B) magnification. The 5-nm gold particles (visible as black spots) distributed on the support grid around the cells suggests that chaperonins were released during sample preparation, but the abundance of gold particles remaining associated with cells indicates that a large number of chaperonins remained associated with an insoluble matrix. The pattern of gold particles in some regions is suggestive of filaments. Uranyl acetate-stained filaments, such as those shown in Fig. 4, were not seen in samples prepared for ImmunoGold labeling, therefore uranyl acetate staining was omitted to visualize better the distribution of gold particles.
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