A membrane-bound archaeal Lon protease displays ATP-independent proteolytic activity towards unfolded proteins and ATP-dependent activity for folded proteins

Abstract

In contrast to the eucaryal 26S proteasome and the bacterial ATP-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain, Archae. We cloned a gene homologous to ATP-dependent Lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal Lon. Lon from Thermococcus kodakaraensis KOD1 (Lon(Tk)) is a 70-kDa protein with an N-terminal ATPase domain belonging to the AAA(+) superfamily and a C-terminal protease domain including a putative catalytic triad. Interestingly, a secondary structure prediction suggested the presence of two transmembrane helices within the ATPase domain and Western blot analysis using specific antiserum against the recombinant protein clearly indicated that Lon(Tk) was actually a membrane-bound protein. The recombinant Lon(Tk) possessed thermostable ATPase activity and peptide cleavage activity toward fluorogenic peptides with optimum temperatures of 95 and 70 degrees C, respectively. Unlike the enzyme from Escherichia coli, we found that Lon(Tk) showed higher peptide cleavage activity in the absence of ATP than it did in the presence of ATP. When three kinds of proteins with different thermostabilities were examined as substrates, it was found that Lon(Tk) required ATP for degradation of folded proteins, probably due to a chaperone-like function of the ATPase domain, along with ATP hydrolysis. In contrast, Lon(Tk) degraded unfolded proteins in an ATP-independent manner, suggesting a mode of action in Lon(Tk) different from that of its bacterial counterpart.

FIG. 1.

(A) Nucleotide and deduced amino acid sequence of Lon_Tk. The sequences with underlined and highlighted characters represent conserved motifs in the AAA⁺ modules and putative transmembrane domains, respectively. The asterisks indicate putative catalytic triad in the protease domain. (B) Comparison of primary structures between Lon_Tk and Lon_Ec.

FIG. 2.

(A) SDS-PAGE gel of purified Lon_Tk from recombinant E. coli. Arrowheads show the four minor bands in the preparation. Lane M, molecular marker. (B) Western blot analysis of Lon_Tk in crude extract, cytosol, and membrane fractions from T. kodakaraensis KOD1 grown on 1% pyruvate and 0.1% elemental sulfur. Numbers on the left side of the panel are molecular masses in kDa.

FIG. 3.

(A) Time courses of hydrolysis of fluorogenic peptide by Lon_Tk. The reaction was carried out in 50 mM Tris-HCl buffer (pH 8.0) containing 5 μg of Lon_Tk and 0.3 mM Glt-AAF-MNA at 75°C. ATP and/or MgCl₂ was added to the mixture as follows: 10 mM MgCl₂ (open circle); 4 mM ATP and 10 mM MgCl₂ (open triangle); no addition (filled circle); or 4 mM ATP (filled triangle). (B) Effects of temperature on peptide cleavage (open circle) and ATPase (filled circle) activities of Lon_Tk. The peptide cleavage assay was performed using Glt-AAF-MNA as a substrate. (C) Effects of ATP (open circle) or AMP-PNP (filled circle) concentration on peptide cleavage activity of Lon_Tk.

FIG. 4.

Proteolytic properties of Lon_Tk with different protein substrates. (A, C, and E) CD spectra of α-casein, hemoglobin A, and Rubisco_Tk at 37°C (gray line) and 70°C (black line), respectively. (B, D, and F) Degradation of α-casein, hemoglobin A, and Rubisco_Tk, respectively, by Lon_Tk. The reaction was carried out at 37 or 70°C with or without the addition of ATP, as indicated in each panel. The mixtures at each reaction time were analyzed by SDS-PAGE as described in Materials and Methods.

FIG. 5.

Degradation of Rubisco_Tk by Lon_Tk with different ATP concentrations (A) and with a nonhydrolyzable analog of ATP, AMP-PNP (B), respectively. The reaction was carried out at 70°C with various concentrations of ATP (0 to 16 mM) for 10 h (A) or with 1 mM AMP-PNP for different reaction periods (B), as indicated in each panel. The mixtures were analyzed by SDS-PAGE as described in Materials and Methods.

FIG. 6.

Schematic model of the function of Lon_Tk in an archaeal membrane in the absence of ATP (A) and in the presence of ATP (B).