A Membrane-Bound Archaeal Lon Protease Displays ATP-Independent Proteolytic Activity towards Unfolded Proteins and ATP-Dependent Activity for Folded Proteins

In contrast to the eucaryal 26S proteasome and the bacterialATP-dependent proteases, little is known about the energy-dependentproteolysis in members of the third domain, Archaea. We cloneda gene homologous to ATP-dependent Lon protease from a hyperthermophilicarchaeon and observed the unique properties of the archaealLon. Lon from Thermococcus kodakaraensis KOD1 (Lon_Tk) is a 70-kDaprotein with an N-terminal ATPase domain belonging to the AAA⁺superfamily and a C-terminal protease domain including a putativecatalytic triad. Interestingly, a secondary structure predictionsuggested the presence of two transmembrane helices within theATPase domain and Western blot analysis using specific antiserumagainst the recombinant protein clearly indicated that Lon_Tkwas actually a membrane-bound protein. The recombinant Lon_Tkpossessed thermostable ATPase activity and peptide cleavageactivity toward fluorogenic peptides with optimum temperaturesof 95 and 70°C, respectively. Unlike the enzyme from Escherichiacoli, we found that Lon_Tk showed higher peptide cleavage activityin the absence of ATP than it did in the presence of ATP. Whenthree kinds of proteins with different thermostabilities wereexamined as substrates, it was found that Lon_Tk required ATPfor degradation of folded proteins, probably due to a chaperone-likefunction of the ATPase domain, along with ATP hydrolysis. Incontrast, Lon_Tk degraded unfolded proteins in an ATP-independentmanner, suggesting a mode of action in Lon_Tk different fromthat of its bacterial counterpart.

INTRODUCTION

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Introduction

Energy-dependent protein degradation in viable cells plays severalimportant roles in the rapid turnover of short-lived regulatoryproteins and in the quality control of intracellular proteinsby eliminating damaged or denatured proteins. In eukaryoticcells, target proteins covalently modified with ubiquitin aredegraded by the multimeric 26S proteasome. Bacterial cells possessa number of ATP-dependent proteases, for instance, Lon, ClpAP,ClpXP, HslUV (ClpYQ), and FtsH in Escherichia coli (13, 14).They processively degrade proteins into short oligopeptidesin a highly coupled manner with ATP hydrolysis, and their functionallows cells to achieve balanced growth and respond to externalstress.

Lon protease was the first ATP-dependent protease to be described(44). Lon from E. coli (Lon_Ec) is an 87-kDa protein consistingof N-terminal ATPase and C-terminal protease domains on a singlepolypeptide (5, 11) and forms a homooligomer (a homotetrameror homooctamer) of identical subunits (6, 11). Previous studiesdemonstrated that a variety of temperature-sensitive mutationsin E. coli could be suppressed by inactivation of the lon gene(16, 17). Lon_Ec participates in the rapid degradation of abnormalproteins in cooperation with the heat shock chaperone systemDnaK/DnaJ/GrpE (21, 43). In addition, it has been known thatLon proteases are capable of recognizing and degrading specificprotein substrates, such as UmuDC (10), SulA (31), RcsA, (7,47), CcdA (48), and so on. Lon_Ec shows little or no peptidecleavage activity in the absence of nucleotides; however, interestingly,it has been suggested that ATP binding, not hydrolysis, at thehigh-affinity sites might induce some conformational changeof Lon_Ec to the open (active) state that allows cleavage ofpeptide bonds (11, 12, 29).

ClpAP, ClpXP, and HslUV (ClpYQ) represent another type of ATP-dependentproteases with a heteromeric structure. They are comprised oftwo distinct subunits of ATPase (ClpA, ClpX, and HslU) and protease(ClpP and HslV) (13, 14). In the recently determined crystalstructure of HslUV, two hexameric rings of HslU were found tobe stacked to both ends of an internal chamber composed of twohexameric rings of the proteolytic core, HslV, forming a structureresembling the eukaryotic 26S proteasome (4, 41, 50). ClpAPis supposed to have a common molecular architecture, exceptfor a tetradecameric proteolytic chamber of ClpP with sevenfoldsymmetry (49). It has been revealed that the hexameric ringof ClpA is self assembled when ATP is bound (22).

The ATPases in the Lon and Clp proteases share structural andmechanistic features with other chaperone-like ATPases thatassist in the assembly, operation, and disassembly of proteincomplexes, and this sequence set is assigned to the AAA⁺ superfamily(34). ATPases in ATP-dependent proteases also act as molecularchaperones, and recent studies have led to considerable progressin understanding the role of ATPase subunits in the proteolysisby ClpAP and ClpXP (15, 55). That is, ClpA and ClpX ATPasesfunction to unfold protein structures, to bind unfolded proteins,and to translocate unfolded proteins to the proteolytic chamber,coupled with ATP hydrolysis (19, 20, 39). Likewise, a chaperone-likeactivity of the ATPase domain of Lon_Ec has been suggested tofunction during the ATP-dependent unfolding of a specific substrate,CcdA (48). A model for the protein unfolding-coupled translocationmechanism has been proposed from the crystal structure of theHslUV complex (50). In addition, proteolytically inactive Lonof yeast mitochondria could complement a mutation of host cellslacking functional assembly of mitochondrial protein complexes,suggesting a chaperone activity of ATPase domain in yeast Lon(35).

In contrast to members of the domains Bacteria and Eucarya,little is known about the energy-dependent proteolysis systemin members of the third domain, Archaea. Complete genome sequenceshave revealed that archaeal genomes also encode putative orthologsof 20S proteasome and proteasome-activating nucleotidases, aswell as open reading frames that share significant identitywith Lon proteases from bacterial cytosol and eucaryal mitochondria.clp and other ATP-dependent protease genes are not found inarchaeal genomes, except for the thermophilic methanogen Methanobacteriumthermautotrophicum, which encodes a Clp-like ATPase (MTH284)but not a Clp-like protease in the genome. Although there havebeen some reports concerning archaeal proteasomes (27, 38, 56),no investigation has been made for the ATP-dependent proteaseLon from archaea.

Thermococcus kodakaraensis (previously called Pyrococcus kodakaraensis)strain KOD1 is a hyperthermophilic archaeon isolated from ageothermal spring at a wharf on Kodakara Island, Kagoshima,Japan (32). Various thermostable enzymes from this archaeon,including DNA polymerase (18, 45), DNA ligase (33), ribulose1,5-bisphosphate carboxylase/oxygenase (8, 24, 26), and so onhave been characterized. Many of the proteins show distinctstructural and catalytic features from bacterial and eucaryalcounterparts. Here, we have characterized an archaeal Lon fromthis hyperthermophile and report the unique properties of thearchaeal Lon protease.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains, plasmids, and media. E. coli TG-1 and DH5 ${alpha}$ and plasmid pUC118 were used for DNA manipulationand sequencing. E. coli BL21CodonPlus(DE3)-RIL cells (Stratagene,La Jolla, Calif.) and plasmids derived from pET-21a(+) or pET-16b(+)(Novagen, Madison, Wis.) were used for gene expression. E. colistrains were cultivated in Luria-Bertani medium at 37°C,and ampicillin was added to the medium at a final concentrationof 50 µg/ml, when needed.

DNA manipulation and sequencing. DNA manipulations were performed by standard procedures, asdescribed by Sambrook and Russell (36). Restriction enzymesand other modifying enzymes were purchased from Takara Shuzo(Kyoto, Japan) or Toyobo (Osaka, Japan). Small-scale preparationof plasmid DNA from E. coli cells was performed with a plasmidmini kit (Qiagen, Hilden, Germany). DNA sequencing was performedwith the ABI PRISM kit and model 310 capillary DNA sequencer(Applied Biosystems, Foster City, Calif.). Nucleotide and aminoacid sequences were analyzed with GENETYX software (SoftwareDevelopment, Tokyo, Japan).

Cloning and expression of lon_Tk. The lon gene from T. kodakaraensis KOD1 (lon_Tk) was identifiedby random sequencing of a phage library of T. kodakaraensisKOD1 genomic DNA, and the nucleotide sequence was determinedwith the phage DNA as a template. The full-length lon_Tk flankedby the NdeI and BamHI sites was amplified by PCR with KOD1 genomicDNA and two primers (sense [5'-ATGCATCATATGGACGAGGAGTCCACCA-3']and antisense [5'-ATGCATGGATCCAGTTAGGAAGACCCGTAAAGCGGAAG-3'];the italicized sequences indicate the NdeI site in the senseprimer and the BamHI site in the antisense primer). The amplifiedDNA fragment was digested with NdeI and BamHI after confirmationof the sequence. The fragment was then ligated with the correspondingsites of plasmid pET-21a(+) to obtain pETlon for the productionof recombinant Lon protease from T. kodakaraensis KOD1 (Lon_Tk)with the native structure. E. coli BL21CodonPlus(DE3)-RIL harboringpETlon was induced for overexpression with 1 mM isopropyl-ß-D-thiogalactopyranoside(IPTG) at the mid-exponential growth phase and incubated for4 h at 37°C. The cells were harvested by centrifugation(7,000 x g for 10 min at 4°C), washed with 50 mM Tris-HClbuffer (pH 8.0), and then resuspended in the same buffer. Thecells were disrupted by sonication and centrifuged (15,000 xg for 30 min at 4°C). The resulting supernatant was incubatedat 70°C for 30 min, followed by centrifugation (15,000 xg for 15 min at 4°C). The precipitated fraction was treatedwith 1.5% Triton X-100 in the Tris buffer at 4°C for 2 hand then centrifuged (15,000 x g for 15 min at 4°C) to obtainsolubilized recombinant Lon_Tk. The supernatant was applied toa ceramic hydroxyapatite CHT5 column (Bio-Rad, Hercules, Calif.)preequilibrated with 5 mM sodium phosphate buffer (pH 7.0).Lon_Tk was eluted by a linear gradient of sodium phosphate buffer(5 to 500 mM) by using a protein purification apparatus (ÄKTAexplorer 10S; Amersham Pharmacia Biotech, Uppsala, Sweden) witha flow rate of 2.0 ml/min. The peak fractions were concentratedby using Centricon-100 (Millipore, Bedford, Mass.), and thesample was applied to a Superdex 200 high resolution 10/30 column(Amersham Pharmacia Biotech) preequilibrated with 50 mM Tris-HCl(pH 7.5) containing 150 mM NaCl with a flow rate of 0.2 ml/min.

In order to purify Lon_Tk without using Triton X-100, a recombinantLon flanked with a His₁₀ tag at the N terminus (Lon_TkNHis₁₀)was expressed and purified by Ni²⁺ affinity chromatography.An expression plasmid, pETlonNHis, was constructed by insertingthe NdeI-BamHI fragment of lon_Tk into pET-16b(+). E. coli BL21CodonPlus(DE3)-RILcells were then transformed with pETlonNHis and were inducedand disrupted as described above. The soluble protein fractionwas applied to a Ni²⁺ affinity column (HisBind purificationkit; Novagen) and washed with 200 mM imidazole in 160 mM Tris-HClbuffer (pH 7.9) containing 4 M NaCl, and Lon_TkNHis₁₀ was elutedwith 1 M imidazole in the buffer.

The protein concentration was determined according to the methodof Bradford (4a) (Bio-Rad protein assay) with bovine serum albuminas a standard. The protein purity was examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysisperformed according to the standard procedure. The N-terminalamino acid sequences of purified proteins were determined bya protein sequencer (cLC model 491; Applied Biosystems).

Enzyme assays. Peptide cleavage activity was assayed using the fluorogenicpeptides glutaryl-Ala-Ala-Phe-4-methoxy-ß-naphthylamide(Glt-AAF-MNA) or succinyl-Phe-Leu-Phe-4-methoxy-ß-naphthylamide(Bachem, Bubendorf, Switzerland) (52). The reaction mixturewas composed of 0.3 mM fluorogenic peptide, 1 or 4 mM ATP, 10mM MgCl₂, and Lon_Tk (5 µg) in 500 µl of 50 mM 2-(cyclohexylamino)ethanesulfonicacid (CHES) buffer (pH 9.0). The mixture was incubated at 70°Cfor 60 min, and the increase in fluorescence (350 nm, excitation;440 nm, emission) was monitored by spectrofluorometer (modelF-2000; Hitachi, Tokyo, Japan).

ATPase activity was assayed by determining P_i liberated fromATP. Lon_Tk (2.5 µg) was incubated with 4 mM ATP and 10mM MgCl₂ in 100 µl of 50 mM CHES buffer (pH 9.0) at 70°Cfor 15 min. Free P_i was determined by the procedure describedby Black and Jones (3). The background levels of hydrolysis(no enzyme) were subtracted in each assay.

Proteolytic properties of Lon_Tk towards ${alpha}$ -casein from bovinemilk (Sigma, St. Louis, Mo.), hemoglobin A from human (Sigma),and ribulose-1,5-bisphosphate carboxylase/oxygenase from T.kodakaraensis KOD1 (Rubisco_Tk) (8) were analyzed by SDS-PAGE.Substrate protein (3.2 µg) was incubated with Lon_Tk (2µg)-1 mM ATP-10 mM MgCl₂ in 100 µl of 50 mM CHESbuffer (pH 9.0) at 37°C for the appropriate amount of time.The mixtures were concentrated to approximately 10 µlunder reduced pressure, applied to SDS-PAGE, and then stainedwith Coomassie brilliant blue R-250 (Bio-Rad). The experimentswere also performed at 70°C with slight modification. Inorder to avoid the complete consumption of ATP in the mixture,the amounts of protein substrate and Lon_Tk were reduced to 0.5and 0.2 µg, respectively, and Bio-Rad silver stain wasapplied for visualization of protein bands. Circular dichroism(CD) spectra of protein substrates were obtained in 10 mM Tris-HClbuffer (pH 8.3) containing 10 mM MgCl₂ by using a spectropolarimeter(model J-820; JASCO, Tokyo, Japan). Matrix-assisted laser desorptionionization-time of flight mass spectroscopic and electron-sprayionization-time of flight mass spectroscopy/mass spectroscopicanalyses of the degradation products from enzymatically dephosphorylated ${alpha}$ -casein (Sigma) were carried out with AXIMA-CFR (Shimadzu, Kyoto,Japan) and API QSTAR (Applied Biosystems), respectively, afterdesalting the mixture by using the ZipTip µC₁₈ column(Millipore).

Western blot analysis. T. kodakaraensis KOD1 was cultivated in artificial seawater(Marine Art SF; Senjyu Seiyaku, Osaka, Japan) containing 0.5%tryptone and 0.5% yeast extract. The cells grown on 1% pyruvateor 0.1% elemental sulfur in the medium at 85°C for 12 hwere harvested, washed with 0.1 M potassium phosphate buffer(pH 7.0) containing 0.4 M NaCl, and then disrupted by Frenchpress (96 MPa). The resulting cell extract was ultracentrifuged(110,000 x g for 2 h at 4°C) to separate the cytosol andmembrane fractions. Each fraction (20 µg of protein) wassubjected to SDS-PAGE and followed by Western blot analysisusing specific antiserum (rabbit) against the recombinant Lon_Tk.A protein A-peroxidase conjugate was used to visualize the specificproteins together with 4-chloro-1-naphthol and hydroxyperoxide.

Nucleotide sequence accession number. The nucleotide sequence data reported here will appear in theEMBL/GenBank/DDBJ nucleotide sequence databases under accessionnumber AB066562.

RESULTS

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Results

Primary structure of Lon_Tk. We found a phage clone harboring a gene homologous to knownlon genes through random sequencing of the T. kodakaraensisKOD1 genomic DNA library. The full-length lon_Tk was subcloned,and the primary structure was determined, as shown in Fig. 1.lon_Tk (1,905 bp) encoded a protein consisting of 635 amino acids(aa) with a molecular mass of 70 kDa, which was smaller thanthe 87-kDa Lon_Ec (5, 11). Lon_Tk possessed a two-domain structurelike other Lon proteases. The N-terminal region was an ATPasedomain belonging to the AAA⁺ superfamily with Walker A and B,sensor 1 and 2, and several replication factor C box motifs,as previously described by Neuwald et al. (34). Nevertheless,the overall identity of the ATPase domain of Lon_Tk was verylow compared to the values for Lon_Ec (20%) and other bacterialand eucaryal Lon proteases. Interestingly, two transmembranehelices could be identified at positions 128 to 150 and 160to 182, which were between the Walker A and B motifs in theATPase domain. The protease domain of Lon_Tk, comprising theC-terminal region, showed 37% identity to that of Lon_Ec, andSer679, which functions as a nucleophile during peptide bondcleavage (2, 9), was also conserved in Lon_Tk at position 521.In Lon_Ec, His665, His667, and Asp676 have been found to be importantresidues and were supposed to form a catalytic triad togetherwith Ser679, as in the case of classical serine proteases (42).The putative counterparts for His665 and Asp676 were conservedin Lon_Tk as His507 and Glu518, respectively, but His667 wasreplaced by Gln at the corresponding position in Lon_Tk.

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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.

Recent genome analyses showed the presence of lon-homologousgenes in other archaea, namely, Pyrococcus abyssi (PAB1313),Pyrococcus horikoshii (PH0452), Archaeoglobus fulgidus (AF0364),Methanococcus jannaschii (MJ1417), M. thermautotrophicum (MTH785),Thermoplasma acidophilum (Ta1081), and Halobacterium sp. (VNG0303G).The deduced amino acid sequences of archaeal Lon showed highidentities to Lon_Tk (50 to 87%) except for that from M. jannaschii(36%). In particular, those from Thermococcales were quite similarto Lon_Tk (86 to 87%), although putative lon genes from P. abyssiand P. horikoshii were divided by intein sequences. In contrast,similarities were lower between Lon_Tk and bacterial orthologs,even from hyperthermophilic Thermotoga maritima (TM1633) (24%)and extremely thermophilic Thermus thermophilus (51) (23%).

Expression and purification of recombinant Lon_Tk. In order to investigate the catalytic properties of Lon_Tk, weconstructed an expression plasmid, pETlon, in which lon_Tk wasoriented downstream of the T7 promoter and the designed ribosomebinding site of pET-21a(+). E. coli BL21CodonPlus(DE3)-RIL wasthen transformed with pETlon, and crude Lon_Tk was obtained asa soluble protein after 0.1 mM IPTG induction. There was noinfluence of lon_Tk expression on the growth of the transformant.The crude soluble fraction was then heat treated at 70°Cfor 30 min to precipitate the proteins from the host cell. Thermostableenzymes derived from thermophiles generally maintained theirsolubility even at high temperatures so that those expressedin E. coli could effectively be separated from host-derivedproteins by heat treatment. However, in the case of Lon_Tk, therecombinant protein precipitated and was found in the insolubleprotein fraction after the heat treatment. This was probablydue to the hydrophobic nature derived from the putative transmembranesequences, since addition of Triton X-100 to the precipitatedfraction led to efficient solubilization of an active protein.The solubilized Lon_Tk was further purified by hydroxyapatitechromatography, followed by gel filtration chromatography. SDS-PAGEanalysis indicated the existence of one major band of 70 kDatogether with four faint bands with apparent molecular massesof 64, 50, 38, and 36 kDa (Fig. 2A). The 70-kDa major proteinagreed with the predicted molecular mass of Lon_Tk, and its N-terminalamino acid sequence was identical to the deduced sequence ofLon_Tk. N-terminal sequences of the minor protein bands of 64,50, and 38 kDa were also identical to that of Lon_Tk, and thatof the 36-kDa protein corresponded to an internal sequence ofLon_Tk from aa 324. These facts indicated that these minor proteinswere formed by the partial proteolysis of Lon_Tk during the expressionand purification steps or by the autolysis of Lon_Tk. Therefore,the active fraction after gel filtration was used for furtherbiochemical characterization. We also attempted to obtain recombinantLon_Tk without using any detergent during the purification steps.For this purpose, a plasmid for recombinant Lon_Tk with a His₁₀tag at the N terminus (Lon_TkNHis₁₀) was constructed. Lon_TkNHis₁₀was expressed in E. coli and successfully purified by Ni²⁺ affinitychromatography, although the yield was much lower than thatfor Lon_Tk with the native primary structure described above.

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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.

The native molecular mass of Lon_Tk could not be estimated bygel filtration chromatography because the recombinant Lon_Tkwas eluted from an exclusion volume of the column. This mightbe due to incorporation of the recombinant protein within micellesof the detergent used for the solubilization. However, thislarge molecular mass was still observed in the case of Lon_TkNHis₁₀purified without the use of any detergents, suggesting an intrinsicproperty of recombinant Lon_Tk to form a highly oligomeric structure.

Subcellular localization of Lon_Tk in T. kodakaraensis KOD1. We examined the localization of Lon_Tk carrying two possibletransmembrane sequences in the cells of T. kodakaraensis KOD1.The cytosol and membrane fractions from T. kodakaraensis KOD1were separated by ultracentrifugation and then subjected toWestern blot analysis with specific antiserum against the recombinantLon_Tk, as represented in Fig. 2B. The results clearly indicatethat Lon_Tk was located in the membrane fractions while no signalwas detected in the cytosol fractions from the cells grown onboth pyruvate (fermentation) and elemental sulfur (sulfur respiration).Lon_Tk is actually a membrane-bound protein, which is a significantdifference between it and its bacterial and eucaryal counterparts.

Peptide cleavage activity of Lon_Tk. Peptide cleavage activity of Lon_Tk was determined by using Glt-AAF-MNA(52), which has often been used as a substrate for various Lonproteases. When Lon_Tk was incubated with Glt-AAF-MNA in thepresence of ATP and Mg²⁺, a time-dependent increase in fluorescencedue to hydrolysis of the fluorogenic peptide at a high temperature(75°C) was observed (Fig. 3A). Surprisingly, we furtherfound that Lon_Tk exhibited much higher activity when ATP wasomitted from the reaction mixture, as shown in Fig. 3A. Thehigh ATP-independent activity of Lon_Tk is an unprecedented featureof Lon proteases. Lon_Tk exhibited no cleavage activity withoutMg²⁺, regardless of the presence or the absence of ATP. Theoptimum pH and temperature for the ATP-independent activityof Lon_Tk were determined to be 9.0 and 70°C (Fig. 3B), respectively.Under the optimum condition, Lon_Tk showed a specific activityof 44 ± 9.7 pmol of MNA/µg of protein/h towardGlt-AAF-MNA in the absence of ATP while that in the presenceof 1 mM ATP showed an activity of 15 ± 5.1 pmol of MNA/µgof protein/h. Lon_TkNHis₁₀ also showed higher peptide cleavageactivity without ATP than with ATP (data not shown), which rulesout the possibility that the ATP-independent activity was anartifact derived from the influence of detergent used in thepurification procedure of Lon_Tk.

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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.

Succinyl-Phe-Leu-Phe-4-methoxy-ß-naphthylamide (52)served as a better substrate for Lon_Tk, with a 5.7-fold higherdegradation rate than that toward Glt-AAF-MNA, suggesting thatLon_Tk preferred hydrophobic substrates. Lon_Tk showed the peptidecleavage activity in the presence of various nucleoside triphosphates;however, the highest level of activity was obtained when nonucleotide was added to the mixture, as shown in Table 1. Thesenucleotides seemed to inhibit the activity rather than supportit. ADP, a potent inhibitor for Lon_Ec, also inhibited the activityof Lon_Tk, but the enzyme retained 57% activity in comparisonto the condition with the addition of ATP. The activity withthe nonhydrolyzable analog of ATP, 5'-adenylylimidodiphosphate(AMP-PNP), was similar to that with ATP, as was the case forLon_Ec. Increase in ATP or AMP-PNP concentration resulted indecrease in the activity, and the activity reached half at 1mM ATP or AMP-PNP (Fig. 3C). These results suggested the inhibitionby nucleotide binding and were quite distinct from those forLon_Ec, for which activity elevated with the increase of ATPconcentration from 10^-4 to 10^-2 mM (12). Lon_Tk strictly requireddivalent cations for the activity, regardless of the presenceor absence of ATP (Table 1). Interestingly, Ni²⁺ served as acofactor better than Mg²⁺ and Ca²⁺ could replace Mg²⁺ to a similarextent. Mn²⁺ and Co²⁺ could also support the hydrolysis, althoughthey were less effective than Mg²⁺, Ni²⁺, or Ca²⁺.

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TABLE 1. Relative peptide cleavage activity of Lon_Tk with different nucleotides or divalent cations

ATPase activity of Lon_Tk. Even though Lon_Tk possessed ATP-independent peptide cleavageactivity, the enzyme exhibited inherent ATPase activity as inother Lon proteases. We confirmed formation of ADP from [ ${alpha}$ -³²P]ATPby thin-layer chromatography analysis and did not observe AMPformation (data not shown). The optimum pH and temperature were9.0 and 95°C (Fig. 3B), respectively, and the specific activitywas 7.0 ± 0.6 x 10⁴ pmol of P_i/µg of protein/hunder the optimum condition, which was 3 orders of magnitudehigher than that for peptide cleavage activity. The ATPase activityof Lon_Tk was efficient and highly thermostable. The activitywas strictly dependent on divalent cations such as Mg²⁺, andother divalent cations (Ni²⁺, Ca²⁺, Mn²⁺, and Co²⁺) could support43 to 59% activity of that with Mg²⁺ (Table 2). UTP could beaccepted as a substrate with a hydrolysis rate that was 21%of that for ATP. It is interesting that CTP and dTTP, quitepoor substrates for Lon_Tk, seemed to inhibit the peptide cleavageactivity less than other nucleotides.

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TABLE 2. Relative hydrolysis rates of different nucleotides by Lon_Tk and effects of divalent cations

Previous studies have indicated a regulatory feature of Lonproteases through stimulation of both ATPase and peptide cleavageactivities by interaction with protein substrates at an allostericsite distinct from the proteolytic site. ${alpha}$ -Casein stimulatedboth the peptide cleavage and ATPase activities of Lon_Ec asmuch as 10-fold and 2.8-fold, respectively (30, 53). In orderto examine the allosteric effects of Lon_Tk, ${alpha}$ -casein was addedto the reaction mixture because ${alpha}$ -casein is a good substratenot only for Lon_Ec but also for Lon_Tk (see below). Without ATP,peptide cleavage activity against Glt-AAF-MNA in the presenceof ${alpha}$ -casein was 103% of that in the absence of ${alpha}$ -casein. In thepresence of ATP, the allosteric effects were slightly largerin the peptide cleavage and ATPase activities, which were increasedto 113 and 133%, respectively, by the addition of ${alpha}$ -casein. Lon_Tkshowed weak allosteric activation when ATP was present, butthe effects were not so striking as in those of Lon_Ec.

Proteolytic property of Lon_Tk. We further examined the proteolytic properties of Lon_Tk towardsthree protein substrates with different thermostabilities. ${alpha}$ -Caseinpossessed no specific structure, and the CD spectra of ${alpha}$ -caseinindeed demonstrated that this protein was unfolded without anysecondary structure at different temperatures (37 and 70°C),as shown in Fig. 4A. In SDS-PAGE analyses, the band correspondingto ${alpha}$ -casein readily disappeared regardless of the presence orabsence of ATP, both at 37 and 70°C (Fig. 4B). These resultscoincided with the ATP-independent activity of Lon_Tk towardthe fluorogenic peptides. Hemoglobin A from human is an ${alpha}$ ₂ß₂heterotetramer with a helix-rich structure at 37°C, butthe secondary structure was disrupted at 70°C due to thermaldenaturation (Fig. 4C). When hemoglobin A was used as a substrateat 70°C, Lon_Tk could degrade the unfolded hemoglobin A ineither the presence or the absence of ATP (Fig. 4D). In contrast,this protein substrate was degraded only in the presence ofATP at 37°C, the temperature under which hemoglobin A maintainsits original folded structure, and the degradation at the lowtemperature did not occur in the absence of ATP. The third substratewas ribulose-1,5-bisphosphate carboxylase/oxygenase from T.kodakaraensis KOD1 (Rubisco_Tk), composed of 10 subunits witha unique pentagonal structure (8, 24, 26). It was previouslydemonstrated that the optimum temperature of Rubisco_Tk is extremelyhigh (90°C) (8) and that its structure is stable even athigh temperatures (24). Figure 4E shows that Rubisco_Tk maintainsits secondary structure between 37 and 70°C. Despite thelarge decameric structure of Rubisco_Tk, Lon_Tk could degradethe protein in the presence of ATP at 70°C whereas no degradationcould be observed when ATP was absent (Fig. 4 F). In these experiments,15% (0.15 mM) of ATP was confirmed to have remained in the mixtureeven after the reaction at 70°C for 8 h, since we observeda gradual decrease in the rate of ATP hydrolysis during theincubation.

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FIG. 4. Proteolytic properties of Lon_Tk with different protein substrates. (A, C, and E) CD spectra of ${alpha}$ -casein, hemoglobin A, and Rubisco_Tk at 37°C (gray line) and 70°C (black line), respectively. (B, D, and F) Degradation of ${alpha}$ -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.

In contrast to the higher peptide cleavage activity of Lon_Tkat a lower ATP concentration (Fig. 3C), the degradation of Rubisco_Tkby Lon_Tk was maximal at 0.5 mM ATP and the lower ATP concentrationsresulted in less degradation efficiency (Fig. 5A). ATP concentrationshigher than 0.5 mM also decreased the degradation, probablydue to the inhibition by ADP accumulated in the reaction mixture.These results are similar to those for Lon_Ec, in which the optimumATP concentration for protein degradation was 0.6 mM (25). Inaddition, Fig. 5B shows that the degradation was not observedwhen AMP-PNP was added instead of ATP. The results obtainedhere indicated that coupled hydrolysis of ATP was necessaryfor degradation of folded proteins by Lon_Tk, whereas peptideand unfolded protein substrates could be cleaved by Lon_Tk inan ATP-independent manner.

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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.

To clarify the peptide bond specificity of Lon_Tk, identificationof the degradation products from ${alpha}$ -casein (enzymatically dephosphorylated)was examined. After the degradation by Lon_Tk in the presenceof ATP at 70°C for 30 min, the reaction mixture was appliedto matrix-assisted laser desorption ionization-time of flightmass spectroscopic analysis. The molecular masses of the productsranged from 771.5 to 2,461.4 Da (data not shown), indicatingthat Lon_Tk degrades the protein into short peptides (6 to 20aa), like other known ATP-dependent proteases. We further appliedelectron-spray ionization-time of flight mass spectroscopy/massspectroscopic analysis and could determine the sequence of oneproduct [m/z 614.4 (2⁺)] as NPWDQVKRNA, an ${alpha}$ -S2-casein peptideencompassing aa 107 through 116. This result indicated thatLon_Tk cleaved the substrate at the bonds after 106Leu and 116Ala,and these preferences were the same as those reported for Lon_Ec(28).

DISCUSSION

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Discussion

As seen in other Lon proteases from various sources, Lon_Tk possesseda two-domain structure consisting of N-terminal ATPase and C-terminalprotease domains. Although the ATPase domain of Lon_Tk showedlow overall similarity to those of its bacterial and eucaryalcounterparts, it still belonged to the AAA⁺ superfamily, possessingseveral conserved motifs such as the nucleotide-binding WalkerA and B (34). A region including the sensor 2 motif in E. coliLon and Clp proteases has recently been proposed to be a sensorand substrate discrimination domain for recognition and bindingto their specific substrates (40, 54). This region was alsoconserved in Lon_Tk and might be involved in the discriminationof unknown specific substrates in T. kodakaraensis KOD1. Anothercandidate for the discrimination region in Lon_Ec is a predictedcoiled-coil structure in the N-terminal region (approximately310 aa) flanked to the AAA⁺ module (7), but such an additionalsequence was not found in Lon_Tk. One significant differencein Lon_Tk from other known Lon proteases is the presence of twopossible transmembrane regions within the ATPase domain, andindeed, Lon_Tk was revealed to be located in the membrane ofT. kodakaraensis KOD1 (Fig. 2 B). This protein shared considerablyhigh homology to putative Lon proteins identified in archaea,and the presumable transmembrane sequences were seen in allarchaeal Lon. HslU, an ATPase component of another bacterialATP-dependent protease, also has a large intermediate domain(134 aa) between the Walker A and B motifs. These intermediatedomains, supposed to participate in the binding to protein substrates,are extended outward from the complex (41, 50). The common positionof these insertion sequences suggests that the transmembraneregions of archaeal Lon proteases may also project outward fromthe enzyme core, forming structures for anchoring to the membrane.Bacteria also possess a different type of ATP-dependent protease,FtsH, that is a zinc metalloprotease and a membrane-bound proteinanchored at the N-terminal region (37, 46). Although the positionsof transmembrane segments and catalytic mechanisms were differentbetween archaeal Lon and bacterial FtsH, Lon might play importantroles in the quality control of the membranes of archaeal cells,like FtsH in bacterial cells (1, 23). In contrast to the ATPasedomain, the protease domain of Lon_Tk showed relatively highsimilarity to other Lon from bacteria and eucarya.

Beyond our expectation, Lon_Tk exhibited activity to cleave fluorogenicpeptides in the absence of any nucleotides higher than thatin the presence of nucleotides, a remarkable catalytic differencebetween Lon_Tk and other known Lon proteases. The peptide cleavageactivity was maximal when ATP was absent but diminished withincreases in ATP or AMP-PNP concentration. These results demonstratethat Lon_Tk already had an open conformation without nucleotidebinding. Lon_Tk also possessed thermostable ATPase activity,and this activity was much higher than the peptide cleavageactivity of this enzyme. Not only did the ATPase activity ofLon_Tk require divalent cations but so did the ATP-independentpeptide cleavage activity of Lon_Tk, indicating two independentroles for the cations. As previously proposed (11, 29), thesemetal cations must serve as a factor that contributes to proteolyticactivity, in addition to their function as a cofactor for ATPbinding.

We further examined the proteolytic activity of Lon_Tk with threeprotein substrates. Schematic models of the action of Lon_Tkin the archaeal membrane are presented in Fig. 6. Lon_Tk coulddegrade proteins that lost their secondary structures withoutconsumption of ATP (Fig. 6A), while it required ATP for thedegradation of proteins maintaining their original secondarystructures (Fig. 6B). The degradation of folded proteins occurredin an energy-dependent manner, since nonhydrolyzable AMP-PNPcould not support the degradation. It has been recently clarifiedthat ATPase components in ATP-dependent proteases are molecularchaperones functioning in the unfolding of protein structures(15, 19, 20, 39, 48, 55). As for Lon_Tk, ATP hydrolysis was requiredfor the degradation of protein substrates with folded structures,such as hemoglobin at 37°C or Rubisco_Tk. It should be notedthat the in vitro degradation required a long incubation period.Although this low degradation efficiency is probably due tothe fact that they are not native substrates for Lon_Tk, ourresults suggested a specific role for ATP hydrolysis in thedisruption of the structures of folded protein substrates, arole shared by the ATPase components of other ATP-dependentproteases. The nucleotide binding might induce some conformationalchange in Lon_Tk for the degradation of substrate proteins withfolded structures, when intracellular ATP concentration is sufficient.

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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).

On the other hand, Lon_Ec requires ATP hydrolysis for efficientdegradation of ${alpha}$ -casein, a protein without a specific secondarystructure (12), indicating that ATP hydrolysis is still neededfor the translocation of large unfolded proteins to the proteolyticsites. Unlike Lon_Ec, Lon_Tk showed ATP-independent proteolyticactivity against not only peptide substrates but also largeprotein substrates lacking secondary structure (Fig. 4B and D).These facts point out a further unique property of Lon_Tk;that is, the translocation of unfolded polypeptide chains toproteolytic sites did not require ATP hydrolysis. In the crystalstructures of bacterial HslUV (4, 41, 50) and ClpP (49), theentrance pore from the ATPase ring to the proteolytic chamberis very small (<10 Å), possibly leading to the ATPrequirement for translocation of already unfolded substratesthrough the small pore. In contrast, the ATP-independent translocationin Lon_Tk suggested easier access of unfolded proteins to theproteolytic core without energy consumption. One of the possibilitiesaccounting for the higher accessibility might be a larger entrancepore in Lon_Tk than that in Lon_Ec.

We here reported the first characterization of an archaeal Lonprotease. Lon_Tk was a novel membrane-bound protease, and thepresence of putative transmembrane helices is a common featureamong archaeal Lon orthologs. It can be presumed that archaealLon degrades specific short-lived proteins in addition to abnormalproteins, as well as Lon (14) and FtsH (37) from E. coli. Identificationof the specific substrates will help to better understand theimportant roles of Lon in archaeal cells.

FOOTNOTES

* Corresponding author. Mailing address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-5568. Fax: 81-75-753-4703. E-mail: imanaka{at}sbchem.kyoto-u.ac.jp.

REFERENCES

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