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. 2007 Mar;189(5):2039-45.
doi: 10.1128/JB.01454-06. Epub 2006 Dec 22.

An actin homolog of the archaeon Thermoplasma acidophilum that retains the ancient characteristics of eukaryotic actin

Futoshi Hara  1 , Kan Yamashiro, Naoki Nemoto, Yoshinori Ohta, Shin-ichi Yokobori, Takuo Yasunaga, Shin-ichi Hisanaga, Akihiko Yamagishi
Affiliations

An actin homolog of the archaeon Thermoplasma acidophilum that retains the ancient characteristics of eukaryotic actin

Futoshi Hara et al. J Bacteriol. 2007 Mar.

Abstract

Actin, a central component of the eukaryotic cytoskeleton, plays a crucial role in determining cell shape in addition to several other functions. Recently, the structure of the archaeal actin homolog Ta0583, isolated from the archaeon Thermoplasma acidophilum, which lacks a cell wall, was reported by Roeben et al. (J. Mol. Biol. 358:145-156, 2006). Here we show that Ta0583 assembles into bundles of filaments similar to those formed by eukaryotic actin. Specifically, Ta0583 forms a helix with a filament width of 5.5 nm and an axial repeating unit of 5.5 nm, both of which are comparable to those of eukaryotic actin. Eukaryotic actin shows a greater resemblance to Ta0583 than to bacterial MreB and ParM in terms of polymerization characteristics, such as the requirement for Mg(2+), critical concentration, and repeating unit size. Furthermore, phylogenetic analysis also showed a closer relationship between Ta0583 and eukaryotic actin than between MreB or ParM and actin. However, the low specificity of Ta0583 for nucleotide triphosphates indicates that Ta0583 is more primitive than eukaryotic actin. Taken together, our results suggest that Ta0583 retains the ancient characteristics of eukaryotic actin.

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Figures

FIG. 1.
FIG. 1.
Maximum-likelihood tree of actin and MreB homologs rooted by the heat shock protein hsp70. The internal mode indicated by a star has a bootstrap value of 0.766 for monophyly of group A MreB homologs and eukaryotic actin. Archaeal homologs are shown against a green background. Group A and B homologs are surrounded by red and blue lines, respectively. Standard bar represents 0.1 nucleotide change per site. See Table 1 for accession numbers of the sequences used.
FIG. 2.
FIG. 2.
Polymerization of Ta0583 monitored by light scattering. (A) pH dependence. The light-scattering intensity (LSI) is expressed as a percentage of that attained after 10 min under standard conditions. Colors represent pH values as follows: red, pH 4.0; orange, pH 4.5; yellow, pH 5.0; green, pH 5.5; blue, pH 6.0. (B) Temperature dependence. Red, 65°C; orange, 56°C; yellow, 45°C; green, 35°C; sky blue, 25°C; blue, 15°C; purple, 5°C. (C) Effects of cations on the level of polymerization. Yellow, MgCl2; green, CaCl2; red, NaCl; orange, KCl. (D) ATP concentration dependence. Red diamonds, levels (expressed as percentages) attained at 10 min; orange rectangles, increasing rate (per minute). (E) Depolymerization and repolymerization experiment. Initial polymerization (red) was done under standard conditions. After attainment of the plateau, one sample was chilled on ice for 1 h, while another was kept at 56°C; both samples were then incubated again at 56°C. Orange and yellow, second measurements after incubation at 0 and 56°C, respectively. (F) Polymerization activities of Ta0583 mutants. Red, wild type; orange, mutant A; yellow, mutant AB. (G) Protein concentration dependence of LSI. LSI at 10 min after polymerization is plotted against Ta0583 concentration.
FIG. 3.
FIG. 3.
(A to C) Electron micrographs of the negatively stained Ta0583 bundle structure. (A) Typical view of bundle of Ta0583 fibers; (B) branched bundle; (C) filament with shortest width (0.1 mg ml−1). Bars, 10 nm. (D) Computed Fourier transform of Ta0583 fiber. Yellow circles indicate diffraction spots due to layered lines. (E) Polymerization model of T. maritima MreB. Shown is a contact site of MreB fiber. Orange arrows, A mutation points; purple arrows, B mutation points. Cyan, yellow, magenta, and green, domains 1, 2, 3, and 4, respectively.
FIG. 4.
FIG. 4.
Precipitation analysis of Ta0583. Shown are the Ta0583 bands in the supernatant (sup) and the precipitate (ppt) after centrifugation (at 10,000 × g for 10 min at 25°C) and the input sample (no centrifugation) after polymerization of 0.5 mg ml−1 Ta0583 under standard conditions with ATP, UTP, CTP, or GTP or without NTPs for 5 min. The Ta0583 band corresponds to a molecular mass of 39 kDa, which is consistent with the gene sequence.
FIG. 5.
FIG. 5.
Contact site of Ta0583. This figure was made by residue replacement from the Ta0583 crystal structure (25) (Protein Data Bank accession number 2fsj) with PyMOL. Original contact sites are shown in purple. (A) Lys42Glu in mutant A (red) and Asp191/Glu193 in the neighboring subunit. (B) Arg271Glu in mutant AB (red) and Glu232 in the neighboring subunit.
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