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Originally published In Press as doi:10.1074/mcp.M600322-MCP200 on December 6, 2006.
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Molecular & Cellular Proteomics 6:492-502, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Proteomics Analysis of Thermoplasma acidophilum with a Focus on Protein Complexes*,S

Na Sun{ddagger}, Florian Beck{ddagger}, Roland Wilhelm Knispel{ddagger}, Frank Siedler§, Beatrix Scheffer§, Stephan Nickell{ddagger}, Wolfgang Baumeister{ddagger} and István Nagy{ddagger},

From the Departments of {ddagger} Structural Biology and § Membrane Biochemistry, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried bei München, Germany


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Two-dimensional gel electrophoresis (2DE) and MALDI-TOF MS were used to obtain a global view of the cytoplasmic proteins expressed by Thermoplasma acidophilum. In addition, glycerol gradient ultracentrifugation coupled to 2DE-MALDI-TOF MS analysis was used to identify subunits of macromolecular complexes. With the 2DE proteomics approach, over 900 spots were resolved of which 271 proteins were identified. A significant number of these form macromolecular complexes, among them the ribosome, proteasome, and thermosome, which are expressed at high levels. In the glycerol gradient heavy fractions, 10 as yet uncharacterized proteins (besides the well known ribosomal subunits, translation initiation factor eIF-6-related protein, elongation factor 1, and DNA-dependent RNA polymerase) were identified that are putative building blocks of protein complexes. These proteins belong to the categories of hypothetical or conserved hypothetical proteins, and they are present in the cytosol at low concentrations. Although these proteins exhibit homology to known sequences, their structures, subunit compositions, and biological functions are not yet known.

The archaeon Thermoplasma acidophilum is a member of the Euryarchaeota lineage of the Archaea (1). The favored environments of the genus Thermoplasma are microaerobic, thermal water basins with a temperature of approximately 60 °C, pH values of 1–2, and nutrients consisting mainly of peptides (2). T. acidophilum lacks a rigid cell wall and is pleomorphous with cell sizes varying between 0.2 and 2 µm. The cells can grow relatively fast and retain their structural integrity under these rather extreme conditions; at elevated pH values, the plasma membrane is destroyed, causing immediate cell lysis and death (3). T. acidophilum contains a 1.5-Mbp chromosome that has been sequenced; it comprises 1507 ORFs and 1481 protein-encoding genes, among which 29% are similar to proteins of unknown function, and 16% have no significant similarity to any described protein (4). Although many macromolecular assemblies of T. acidophilum like the proteasome, thermosome, valosin-containing protein-like ATPase from T. acidophilum (VAT ATPase),1 and tricorn protease have been studied in great detail, a general overview on the protein expression profile of this extremophile is not available to date.

This work forms part of a project aimed at visualizing the proteome of T. acidophilum and thereby providing a comprehensive cellular atlas of macromolecular complexes. The idea of this approach, termed "visual proteomics" (5) is based on a multistep procedure that comprises the proteomics (native and denaturing) analysis, the creation of a template library, the acquisition of three-dimensional (3D) cellular tomograms, interpretation of the tomograms by a pattern recognition procedure, and finally the generation of a cellular atlas.

Based on the proteomic inventory of T. acidophilum, 3D structural data covering as much as possible of the macromolecular complement are compiled in a template library in the form of density maps. The sources for this structural information are high resolution methods such as x-ray crystallography, NMR, or electron microscopy single particle techniques. Based on the structural signature of the proteins, a pattern recognition algorithm compares the molecular templates with the density maps of cellular tomograms obtained by cryoelectron tomography (cryo-ET). The match for a particular template at a certain position and orientation is measured by 3D cross-correlation (6). Step by step, all molecules found in the proteome can be matched via their structural fingerprint, and a virtual cellular model of the molecules is compiled. Visual proteomics will enable us to determine the spatial relationships of molecular complexes and to analyze their interaction networks in an unperturbed cellular environment.

Complementary to the native proteome analysis aimed at the separation, identification, and structural characterization of complexes, we investigated the T. acidophilum proteome by two denaturing proteomics approaches. First we established a 2DE reference map that gives a global overview on the cytosolic proteins of T. acidophilum expressed under aerobic growth conditions. Based on database searches, we provide a list of those highly expressed proteins that form complexes and for which 3D structures have been solved, therefore enabling these structures to serve as templates in the cryo-ET template matching experiments. The second approach comprises the prefractionation of the soluble proteins prior to 2DE by glycerol density ultracentrifugation and analysis of fractions containing protein complexes over the size of 1 MDa to identify their subunit composition because available information on these proteins of T. acidophilum is scarce.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Growth of T. acidophilum and Sample Preparation—
T. acidophilum stock (1.5 ml) stored at –80 °C was inoculated into 50 ml of T. acidophilum medium preheated to 58 °C in an oil bath (7). The cells were grown at 58 °C and 120 rpm on a rotary shaker until the A540 reached 0.6 (2 days). Then 10 ml of culture were transferred to 500 ml of preheated complete medium. When the culture reached A540 of approximately 0.6, the cells were harvested by centrifugation at 4000 x g for 10 min at 4 °C and washed with MilliQ H2O. The pellet was resuspended in 4 ml of MilliQ H2O containing protease inhibitors. The pH of the suspension was elevated with 1 M unbuffered Tris to pH 8, 0.5 mg of DNase I was added, and the mixture was incubated on ice until cell lysis occurred. The lysate was centrifuged at 20,000 rpm for 30 min at 4 °C in a Beckman L8-M ultracentrifuge (rotor, 70.1 Ti). The supernatant was used immediately or stored at –80 °C until further use. For 2DE, the cell extracts containing 50 µg (silver staining) or 400 µg (Coomassie Blue staining) of protein were combined with 2DE sample buffer according to the GE Healthcare 2DE protocol (2-D electrophoresis using immobilized pH gradients: principles and methods).

Glycerol Density Gradient Ultracentrifugation—
Extracts (4 mg in 200–500 µl of buffer) from four independent T. acidophilum cultures were loaded directly onto 35-ml 10–50% glycerol gradients in 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, and 1 mM ATP. A BioComp Model 117 Gradient Mate (BioComp Instruments) was set to perform a 3-min run at 15 rpm speed and 80° angle to form a continuous glycerol gradient from the overlaid 10 and 50% glycerol solutions. Centrifugation was carried out for 22 h at 25,000 rpm in a Beckman SW 28 swing-out rotor maintained at 4 °C. After centrifugation, the gradients were fractionated into 35 fractions of 1 ml each by injection of 86% glycerol at the bottom of the tube through a density gradient fractionator (Instrumentation Specialties Co.). The fractions, collected from top to bottom, were loaded both on one-dimensional SDS-PAGE gels to check protein distribution and on native gradient gels to check protein size. Fractions of interest (12–13, 17–19, 22–24, and 26–28, over the size of the 20 S proteasome (700 kDa)) were selected, pooled, and precipitated to obtain higher protein concentration. After precipitation with 13% TCA, protein samples were washed and dissolved in 2DE sample buffer.

2DE—
2DE was done according to the GE Healthcare 2DE protocol using pH 3–11 non-linear 18-cm-long IPG strips (GE Healthcare) for first dimension separation and 11% polyacrylamide (PA) gel in Laemmli buffer system (8) for second dimension separation. IEF was performed with a Multiphor II unit (GE Healthcare) at 20 °C using the following voltage profile: linear increase from 0 to 500 V in 1 min, linear increase from 500 to 3500 V in 1.5 h, and 3500 V for 14 h. Total focusing time was about 49,000 V h. The IPG strips were stored at –80 °C if they were not used immediately. Separation in the second dimension was achieved using the Protean II xi cell (Bio-Rad).

After completion, the 2DE proteins were fixed and stained. Colloidal Coomassie Brilliant Blue G250 staining was carried out according to Neuhoff et al. (9). Briefly PA gels were fixed in 12% TCA for 15 min followed by washing steps with several changes of water. The gels were stained with Coomassie Blue G250 staining solution overnight at room temperature, and the excess of stain was washed out with distilled water. Silver staining was carried out according to Mortz et al. (10) with minor modifications. Gels were fixed twice in 50% (v/v) methanol and 12% (v/v) acetic acid for 30 min, washed three times with 50% (v/v) ethanol for 20 min, and soaked for 1 min in 200 mg/liter Na2S2O3. After washing twice with MilliQ H2O for 1 min, gels were stained for 20 min in a freshly made solution containing 2 g/liter AgNO3 and 375 µl/liter formaldehyde, washed briefly with MilliQ H2O, and developed in a solution including 60 g/liter Na2CO3, 5 mg/liter Na2S2O3, and 250 µl/liter formaldehyde until the desired contrast was obtained. This usually took 1–5 min depending on the temperature. Then the staining solution was drained, gels were washed with MilliQ H2O, and the staining was terminated with 5% (v/v) acetic acid for 10 min. The gels were soaked in 1% (v/v) acetic acid and stored at 4 °C.

Protein Digestion and Sample Preparation—
For spot picking, protein digestion, and sample preparation, we used the procedures and equipment of the Proteomics Service Facility of the Max Planck Institute of Biochemistry following the methods published by Tebbe et al. (11).

Protein Identification—
MALDI-TOF MS spectra were recorded automatically using a Reflex III spectrometer (Bruker Daltonics) with an accelerating voltage of 20 kV. The spectra were externally calibrated with an in-house optimized mixture of eight peptides ranging from 1046.54 to 3494.65 Da. Spectra were automatically annotated using the vendor's "Xmass" program package (version 5.1.16), which returns monoisotopic masses. Parameters used for the macro operation were the following: peak picking method, "SNAP;" maxpks, 40; PC, 2.2; goodness, 30; peakdig, 3. An average mass accuracy of 120 ppm was obtained.

Biotools software (Bruker Daltonics) integrating the locally installed Mascot search engine (Matrix Science, London, UK) (12) was used to search the database comprising all ORFs of T. acidophilum (pedant.gsf.de). Search parameters were as follows: one missed cleavage site, ±200 ppm tolerance (to account for reduced mass accuracy of larger peptides), carbamidomethylation of cysteines as fixed modification, and oxidation of methionine as variable modification. Proteins were considered to be identified if the Mascot program returned a Molecular Weight Search (MOWSE) peptide mass database score of at least 44, indicating a probability of less than 5% that the observed match is a random event.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
2DE Reference Map of T. acidophilum Cytoplasmic Proteins—
Most of the T. acidophilum proteins were found in the pH range 4–7, although the theoretical pI values predicted a protein accumulation around pH 8 as well (data not shown). Comparing the number of the detected spots, we found that despite an 8-fold increase in protein concentration loaded on Coomassie-stained gels (600 spots) more protein spots (933 spots) were visualized with silver staining. The greater sensitivity of the silver staining did not result in an increase in the proportion of proteins identified through MALDI-TOF MS with only 442 (34%) of the 1317 excised spots being identified. In contrast, Coomassie Blue G250 stain gave much better MS results with 1235 (75%) of the 1640 excised spots being identified. Altogether 271 proteins were identified of which 178 were found three times or more, 29 were found twice, and 64 were found once (Supplemental Table S1).

The majority of the spots contained only single proteins, but in several cases the MS analysis indicated a mixture of proteins. When second or third hits could not be verified in consecutive experiments, these were omitted from the protein list.

Protein distribution over multiple spots seems to be common for T. acidophilum (Fig. 1). The isoforms of ribonucleotide reductase (Ta1475), thermosome subunits (Ta0980 and Ta1276), Fe-superoxide dismutase (SOD) (Ta0013), translation elongation factor aEF-1 {alpha} chain (Ta0444), succinyl-CoA synthetase {alpha} subunit (Ta1331), peroxiredoxin 1 (Ta0152), VAT ATPase (Ta0840), glutamine synthetase (Ta1498), a conserved hypothetical protein (Ta0085), S-adenosylmethionine (AdoMet) synthetase (Ta0059), and proteasome {alpha} subunit (Ta1288) are indicated. The reason for this isoform distribution is unknown, but nevertheless the theoretical and experimental protein distribution in 2D gels correlated well, thus corroborating the analysis technique (Supplemental Figs. S1 and S2).


Figure 1
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  		FIG. 1. 2DE reference map of cytosolic proteins of T. acidophilum. Protein isoforms were detected by either IEF (Ta0444, Ta1475, etc.) or by size separation (Ta0013). Undissociated protein complexes were also detected (Ta0013). Only the most prominent proteins are annotated on this map.

 
Functional Categories of the Identified Proteins—
An overview on the distribution of the identified proteins in functional categories (based on the Munich Information Center for Protein Sequences (MIPS) database, mips.gsf.de) including a more detailed description of the most prominent proteins is provided. The complex-forming ability of 18 of these proteins is summarized in Table I. Proteins of Coomassie-stained gels were quantified using PDQuest (BioRad) software; however, a precise quantification is difficult due to the presence of protein isoforms (which requires that each of the spots is analyzed and summed) and to highly expressed proteins that exceeded the dynamic range of the staining method (data not shown).


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  		TABLE I List of highly abundant proteins of T. acidophilum

The gene number (ORF), annotation, complex formation, and the molecular mass of the complex are indicated. Complex formation of the protein in other organisms is also considered (Ta, T. acidophilum; Tt, Thermus thermophilus; Mj, Methanococcus jannaschii; Pf, P. furiosus; Psp, Pyrococcussp. strain KOD1; Ap, Aeropyrum pernixK1).

 
A large proportion of the identified proteins can be assigned a metabolic function (43.0%). These include groups of amino acid (15.6%), nucleotide (9.6%), C-compound and carbohydrate (11%), lipid and fatty acid (5.9%), and vitamin and cofactor (5.1%) metabolism. Proteins involved in energy metabolism were the second most abundant group (16.5%) followed by the categories of protein synthesis (10.2%) and protein fate (5.5%). Three groups, "classification still preliminary" (13.9%), the "unclassified proteins" (11%), and proteins of cellular transport (1.5%) were underrepresented compared with the genome annotation data (Table II).


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  		TABLE II Functional distribution of T. acidophilum cytosolic proteins identified from 2D SDS-PA gels

The MIPS database server was used for automated protein categorization. Both proteome (271 identified proteins) and genome data (1481 ORFs) were submitted to the server, and the results are provided in absolute (match) and relative (%) numbers.

 
Metabolism and Energy Metabolism—
Identified proteins of the non-phosphorylating variant of the Entner-Doudoroff (ED) pathway, the Embden-Meyerhof-Parnas pathway and tricarboxylic acid cycle are listed in Supplemental Table S2. The most abundant enzymes of these biochemical pathways were pyruvate:ferredoxin oxidoreductase, malate dehydrogenase, glyceraldehyde dehydrogenase, and succinyl-CoA synthetase. Pyruvate:ferredoxin oxidoreductase ({alpha} subunit, Ta0259; ß subunit, Ta0260) catalyzes the CoA-dependent oxidative decarboxylation of pyruvate (13). Its activity is crucial in energy metabolism, connecting the ED pathway to the tricarboxylic acid cycle (14, 15). Despite formation of supramolecular assemblies, the size of the complex is on the limit of detection by cryo-ET as we found this enzyme in native gradient PA gels at a size of approximately 300 kDa.2

Malate dehydrogenase (Ta0952) catalyzes the conversion of malate to oxaloacetate utilizing the NAD/NADH cofactor system, and it participates in the tricarboxylic acid cycle, pyruvate metabolism, glyoxylate and dicarboxylate metabolism, carbon fixation, and the reductive carboxylate cycle (CO2 fixation) (16, 17). In addition, the cytosolic malate dehydrogenase has an antioxidant function, minimizing H2O2 or {gamma}-radiation-induced damage to cellular DNA, proteins, and lipids (18).

Succinyl-CoA synthetase ({alpha} subunit, Ta1331; ß subunit, Ta1332) is a member of a large family of acyl-CoA synthetases (nucleoside diphosphate-forming), which also includes acetyl-CoA synthase found in many Archaea and lower eukaryotes. The molecular mass of the {alpha} and ß subunits are ~30 and ~40 kDa, respectively (19), and there are fundamental differences in the quaternary structures of the bacterial and mammalian forms of succinyl-CoA synthetases. The Escherichia coli enzyme functions as an {alpha}2ß2 tetramer with no activity associated with a heterodimeric form, whereas the enzyme isolated from pig heart functions only as the {alpha}ß dimer (20). We found that the T. acidophilum counterpart of the succinyl-CoA synthetases has a molecular mass of 500 kDa, making it a good candidate for cryo-ET analysis.2

Glyceraldehyde dehydrogenase (Ta0809) is a key enzyme in the non-phosphorylative ED pathway, converting glyceraldehyde to glycerate (21). Its complex-forming ability is unknown.

Transcription, Translation, and Protein Fate—
We found a large set of proteins belonging to the complexes of exosome, ribosome, and DNA-dependent RNA polymerase that, together with the presence of a great number of proteins taking part in amino acid (65 proteins) or nucleotide (34 proteins) metabolism, indicates that T. acidophilum maintains highly active biochemical pathways to balance between protein, DNA, and RNA degradation and biosynthesis.

Proteins involved in RNA biosynthesis and degradation were well represented; three subunits of the archaeal DNA-dependent RNA polymerase (Ta0391, Ta0392, and Ta1030) were found as well as the TATA box-binding protein (Ta0199). The DNA-directed RNA polymerase consists of 11 subunits, and the molecular mass of the complex is 500 kDa (22). Subunits (Ta1291, Ta1292, Ta1294, Ta0613, and Ta0929) of the putative exosome, a complex of RNases, RNA-binding proteins, and helicases that mediates processing and 3' -> 5' degradation of a variety of RNA species (23) were also identified.

Proteins needed for protein translation were represented by 14 ribosomal subunits, translation factor Ta0302, translation elongation factor aEF-1 {alpha} (Ta0444), GTP-binding protein (Ta1237), translation initiation factors (Ta1212 and Ta0322), and aminoacyl-tRNA synthetases for valine (Ta0040), serine (Ta0468), asparagine (Ta0946), lysine (Ta1163), histidine (Ta0099), and alanine (Ta0499). One of the most abundant proteins was the translation elongation factor aEF-1 {alpha} chain (Ta0444) that can be present in the cytosol either as a free monomer or in a complex with the ribosome (24).

Proteins that belong to the functional category "protein fate" (determining protein folding, modification, destination, and degradation) were also abundant. At least one member of each chaperone class found in T. acidophilum (4) was expressed (Ta0125, glutaredoxin-related protein; Ta0471, small heat-shock protein (hsp20); Ta0840, VAT ATPase; Ta1175, VAT-2 protein; Ta0866, thioredoxin-related protein; Ta0980, thermosome {alpha}-chain; Ta1276, thermosome ß-chain; Ta1011, peptidyl-prolyl cis-trans isomerase-related protein; and Ta1087, probable DnaK-type molecular chaperone). The identified representatives of the cytosolic protease clans were as follows: Ta1490, tricorn core protease; Ta0301, tricorn cofactor F2; Ta1288, proteasome {alpha} subunit; Ta0612, proteasome ß subunit; Ta1439, methionine aminopeptidase I; Ta1037, proline dipeptidase; and Ta0465, Pfpl-related endopeptidase.

The most abundant proteins of protein fate were the thermosome, VAT ATPase, and proteasome. The thermosome is a molecular chaperone composed of two subunits, {alpha} and ß, that are arranged in two stacked eight-membered rings (900 kDa) with a central cavity that provides a sequestered environment for in vivo protein folding (25). VAT ATPase Ta0840 is a hexameric (~500-kDa, 15.5-nm) archaeal member of the Cdc48/p97 family of ATPases associated with a variety of cellular activities (AAA ATPases) (26). It has two ATPase domains and a 185-residue amino-terminal substrate recognition domain, VAT-N. VAT shows activity in protein folding and unfolding and thus shares the common function of these ATPases in disassembly and/or degradation of protein complexes (26). The 20 S proteasome is a macromolecular assembly (700 kDa) designed to confine proteolytic activity to an inner cavity. Access to the central proteolytic nanocompartment is restricted to unfolded proteins, necessitating a functional coupling of the 20 S proteasome to a substrate recognition and unfolding machinery (27). Most of the peptides generated by the proteasome are degraded further to single amino acids that can be used in cell metabolism and for the synthesis of new proteins. In T. acidophilum, the giant tricorn protease (Ta1490) and its interacting factors work downstream of the proteasome, processing the peptides into amino acids (28). However, we found that the quantity of the expressed tricorn protease was much lower than that of the proteasome levels (data not shown).

Abundant Proteins of Amino Acid Metabolism—
Glutamine synthetase (Ta1498) produces glutamine from glutamic acid and ammonia. It forms a dodecameric complex in bacteria that is comprised of two face-to-face hexameric rings forming a cylindrical aqueous channel (29). It was also shown to be functional in a dodecameric form (637 kDa) in the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (30), and we therefore anticipate that it has a similar architecture in T. acidophilum.

Anabolic ornithine carbamoyltransferase (OTCase) (Ta1330) catalyzes the carbamoylation of L-ornithine to form citrulline in the sixth step of the arginine biosynthesis pathway. Most anabolic OTCases form trimers of about 105 kDa. The anabolic OTCase of the hyperthermophilic archaeon Pyrococcus furiosus is a dodecamer composed of four catalytic trimers (420 kDa) (31, 32), making it suitable for cryo-ET analysis.

Ta0085 is a conserved hypothetical protein showing homology to L-alanine-DL-glutamate epimerase and related enzymes of the enolase superfamily. In other organisms, members of this family take part in cell wall and/or outer membrane biogenesis (Kyoto Encyclopedia of Genes and Genomes (KEGG) database, www.genome.ad.jp/kegg). As T. acidophilum is devoid of a cell wall and it bears a special cell envelope, it will be interesting to study the function of this protein.

Vitamin, Cofactor, and Prosthetic Group Metabolism—
Interestingly proteins that catalyze the biosynthesis of coenzymes like folate (Ta0079 and Ta1022) and vitamins B12 (Ta0078, Ta0571, and Ta0652–Ta0660) and B6 (pyridoxine biosynthesis PyroA protein, Ta0522) were highly expressed. Ta0078 is a conserved hypothetical protein that shows homology to CobT. This putative phosphoribosyltransferase plays a central role in the synthesis of {alpha}-ribazole-5'-phosphate, an intermediate for the lower ligand of cobalamin that is essential for de novo cobalamin (vitamin B12) synthesis in bacteria (33). Strikingly the highly abundant enzyme ribonucleotide reductase (Ta1475) that catalyzes the conversion of both purine and pyrimidine nucleotides to deoxynucleotides in all organisms and provides all the monomeric precursors essential for both DNA replication and repair (34) needs B12 to carry out its catalytic functions (35).

The synthesis of numerous biological compounds and the regulation of many metabolic processes require the addition or removal of one-carbon units. Ta0060, Ta0898, Ta1476, and Ta1478 are identified enzymes that take part in the one-carbon pool by means of folate. Tetrahydrofolate coenzymes function as one-carbon unit carriers that are involved in several major cellular processes including the synthesis of purines and thymidylate, amino acid metabolism, pantothenate synthesis, and Met synthesis. Met is the direct precursor of AdoMet, which in turn is the source of methyl units for the synthesis of a myriad of molecules (36). AdoMet synthetase (Ta0059) catalyzes the only known route of AdoMet biosynthesis (37) and therefore plays a central role in the metabolism of all cells. The biological roles of AdoMet include acting as the primary methyl group donor for DNA and protein methylation, as precursor to the polyamines, and as a progenitor of a 5'-deoxyadenosyl radical (38–40).

Cell Rescue and Defense Proteins—
We identified 11 of the annotated 27 cell defense proteins. The most prominent highly expressed members of this group were the antioxidative enzymes SOD (Ta0013), peroxiredoxins (Ta0152, Ta0473, and Ta0954), and the alkyl hydroperoxide reductase subunit F (Ta0125). SOD is a metalloenzyme playing a central role in the defensive system of organisms toward the toxicity of superoxide radicals (41). Peroxiredoxins contain a reactive Cys residue in the conserved region near the amino terminus, and this catalytic Cys residue forms cysteine-sulfenic acid as a reaction intermediate during the reduction of peroxide (42). Ta0125 exhibits homology to alkyl hydroperoxide reductase subunit F (AhpF) that together with AhpC (a peroxiredoxin homologue) catalyzes the NADH-dependent reduction of organic hydroperoxides (or hydrogen peroxide) to their corresponding alcohols and water (43). For the structure, mechanism, and regulation of peroxiredoxins, we refer the reader to a recent review (44).

Putative Complex-forming Proteins Identified by Glycerol Density Gradient Ultracentrifugation Coupled to 2DE-MALDI-TOF MS—
Glycerol density gradient ultracentrifugation coupled to 2DE-MALDI-TOF MS was used for the identification of proteins that most probably are building blocks of macromolecular complexes over the size of 1 MDa. First aliquots of the glycerol gradient fractions were loaded on one-dimensional gels to localize subunits of known macromolecules (proteasome and thermosome) using MS followed by pooling fractions 12–13 (gel A), 17–19 (gel B), 22–24 (gel C), and 26–28 (gel D), respectively, and analysis by 2DE (Fig. 2, A–D).


Figure 2
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  		FIG. 2. 2D display of cytosolic proteins of T. acidophilum from fractions obtained by glycerol gradient ultracentrifugation. Fractions 12–13 (A), 17–19 (B), 22–24 (C), and 26–28 (D) were pooled, respectively, and analyzed by 2DE. The appearance or increasing intensity of a protein spot in gels B, C, and D indicates that the given protein sediments as a subunit of a large complex (marked with rectangles). Putative complex-forming proteins are annotated on gels C and D. Decreasing spot intensity indicates complexes of lower molecular weight, and as representatives of this category peroxiredoxin (Ta0152) and the proteasomal (Ta0612 and Ta1288) and the thermosomal (Ta0980 and Ta1276) subunits are shown (marked with ovals).

 
The appearance or increasing intensity of a protein spot in gels B, C, and D indicated that the protein sedimented as a subunit of a larger complex, whereas we judged that the faded appearance of a protein in these gels indicated a complex of lower molecular weight (i.e. proteasome, thermosome, and peroxiredoxin Ta0152). Putative complex-forming proteins are listed in Table III.


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  		TABLE III List of proteins identified from the heavy glycerol gradient ultracentrifugation fractions, which exhibited increasing intensity in four consecutive gels

The gene number (ORF), protein annotation, function, and comments are indicated.

 
The function and structure of several of them like the ribosome, translation initiation factor eIF-6-related protein, elongation factor 1, the DNA-dependent RNA polymerase, and the pyruvate dehydrogenase complex are well studied. The rest of the proteins belong to the categories of "hypothetical" or "conserved hypothetical proteins." We found by BLAST (basic local alignment search tool) search that Ta0316 is a twitching motility (PilT)-related protein. Reportedly PilT is a large hexameric ATPase that is required for pilin retraction, disassembly, and degradation (45). However, Ta0316 might have other functions as a more detailed sequence analysis using the SMART (Simple Modular Architecture Research Tool) server revealed three conserved domains (smart.embl-heidelberg.de). The amino-terminal sequence encodes for PilT N terminus (PINc) that is predicted to play role in nucleotide binding and is potentially found in RNases. The central part of the protein showed homology to AAA ATPase sequences, and on the carboxyl terminus there was a ribonucleoprotein K homology type 1 domain, a motif found in nucleic acid (mostly RNA)-binding proteins.

Ta0522 showed homology to the pyridoxine biosynthesis PyroA protein. PyroA proteins form a highly conserved protein family with members in the Archaea, Bacteria, and Eukarya, and the pyroA gene product of Aspergillus nidulans is required for the biosynthesis of pyridoxine and resistance to photosensitizers such as methylene blue (46). The other proteins like Ta0078 (CobT homologue), Ta0341 (glycogen-debranching enzyme 1 related protein), Ta0890 (predicted transcriptional regulator), Ta1155 (probable ribonuclease Z), and Ta1201 (probable 3-isopropylmalate dehydrogenase) also exhibited homology to known sequences to some extent, but their 3D structure, quaternary structure, subunit composition, and biological function remain to be investigated.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The work presented here gives an overview on the expressed cytosolic proteins and on the macromolecular complexes of T. acidophilum cultured under aerobic growth conditions at 58 °C, pH 1.5–1.8. We used the 2DE-based protein separation and MALDI-TOF MS protein identification method and Coomassie Blue or silver staining to visualize proteins displayed in denaturing gels. Based on database search, we established a list of proteins that form complexes and for which a 3D structure has been solved, therefore allowing such complexes to serve as templates in cryo-ET pattern recognition. Additionally we coupled 2DE-MALDI-TOF MS to glycerol gradient ultracentrifugation protein separation to identify proteins that are constituents of larger complexes.

The protein separation by 2DE in combination with MALDI-TOF MS protein identification is a common method to investigate the proteome of organisms whose genome sequence is known. It was especially suitable for T. acidophilum as it has a relatively small genome with 1481 protein-coding ORFs, and the number of protein spots that can be analyzed in a 2D gel lies in the range of several thousand. Despite the distribution of many proteins in multiple spots (isoforms), the protein resolution with the chosen IEF strip and second dimensional gel size was high enough to display single proteins (over 900 spots were resolved of which 271 proteins were identified) and to obtain a satisfactory separation of acidic and basic proteins as well. The two staining methods used in our experiments were complementary to each other; with Coomassie G250 we could detect fewer spots, but the protein identification ratio was much higher than that with silver. Coomassie-stained gels were analyzed to distinguish highly and poorly expressed proteins.

The large majority of the identified proteins participate in fundamental biochemical pathways like energy metabolism, energy production, amino acid metabolism, purine and pyrimidine biosynthesis, replication, transcription, translation, RNA degradation, protein degradation, cell membrane biosynthesis, fatty acid metabolism, and cofactor biosynthesis. A proteomics approach on a natural acid mine drainage biofilm community consisting of Leptospirillum and Ferroplasma species (47) showed similar results. Ribosomal proteins (13%), chaperones (11%), thioredoxins (9%), and proteins involved in defense against reactive radical species (8%) were also highly abundant, indicating a lifestyle of permanent struggle against oxidative stress. Additionally proteins involved in amino acid metabolism, translation, energy production and conversion, cell envelope biogenesis, coenzyme metabolism, and protein folding and modification were also abundant.

The high expression level of the proteasomes, chaperones (thermosome, VAT, and DnaK), elongation factors, translation initiation factors, aminoacyl-tRNA synthases, and ribosomes in T. acidophilum cells indicates a high protein turnover rate. This can be due to the production of large amounts of reactive oxygen species and peroxide that can oxidize or otherwise damage cell constituents, mostly proteins, as these are present in the cell in the highest amounts (48). There is an active detoxifying process in the T. acidophilum cells, marked with large quantities of SOD, alkyl hydroperoxide reductase, and three peroxiredoxins, but it is likely that their activity is not satisfactory, and this results in protein, RNA, and DNA damage and consequently high macromolecular turnover. Supporting evidence for the fast RNA and DNA turnover can be the extremely high amount of ribonucleotide reductase that catalyzes the production of desoxyribonucleotides from ribonucleotides. This enzyme needs vitamin B12 for its activity (35), and we found 10 proteins of the B12 biosynthesis pathway indicating that most probably this vitamin is produced de novo.

Koonin et al. (23) described a superoperon of exosomal genes in Archaea that in addition to the predicted exosome components encodes the catalytic subunits of the proteasome, two ribosomal proteins, and a DNA-directed RNA polymerase subunit. These observations suggest that in Archaea a tight functional coupling exists between translation; RNA processing and degradation, apparently mediated by the predicted exosome; and protein degradation, mediated by the proteasome. Although the RNase P subunits are missing in T. acidophilum, we suppose that the remaining expressed exosomal proteins are functional. It will be interesting to study their complex-forming ability and to compare these to the recently solved structure of exosome RNase PH core complex of Sulfolobus solfataricus (49). In contrast to findings concerning the S. solfataricus exosome, we could not confirm co-sedimentation of the T. acidophilum exosomal counterpart with the ribosome as there were no detectable exosomal subunits in the heavy glycerol gradient fractions.

Besides the proteins participating in central biochemical processes, we found that 14% of the proteins belonged to the group of hypothetical/conserved hypothetical proteins. The function of these proteins in T. acidophilum remains elusive. We found several proteins such as a ß-galactosidase homologue (Ta1323) or Ta1060 exhibiting similarity to the bacterial atrazine-degrading chlorohydrolase that might have industrial applicability. Other proteins like Ta0247 (a homologue of carboxysome-forming proteins) and Ta0881 (a carbonate dehydratase homologue that is associated with the carboxysome) were also found. Carboxysomes are polyhedral inclusion bodies present in CO2-fixing microorganisms (50, 51); they have a size of 120 nm and serve to protect ribulose-1,5-bisphosphate carboxylase/oxygenase (52). There is no evidence for the presence of carboxysome or carboxysome-related structures in T. acidophilum; the function(s) of these proteins needs further investigation. We found evidence that a putative glycogen-debranching enzyme was also expressed under the given conditions, and the KI reaction indicated the presence of a starchlike polymer in the crude extract (data not shown), although we could not find a homologue of the glycogen initiation peptide. Glycogen has a globular shape, and it has an average size of 40 nm (53). The electron microscopy analysis of glycogen is available (53), and it can serve as template for our cryo-ET analyses to verify these observations. In the search for candidates for archaeal cytoskeleton, we found that the MreB homologue (Ta0583) and Ta1488 were expressed. The formation of filaments by these proteins has yet to be demonstrated in vitro and in vivo.

In conclusion, the 2DE-MALDI-TOF MS proteomics approach provided information on macromolecular complexes of T. acidophilum. Using the cytoplasmic proteome analysis, we identified abundant complex-forming proteins, the structure, subunit composition, and biological function of which are mostly well studied in T. acidophilum or in other Archaea, whereas the glycerol gradient protein prefractionation resulted in the identification of higher molecular weight complexes expressed at low level that have not been studied in T. acidophilum previously.


   ACKNOWLEDGMENTS
 
We thank Dr. Andrew Leis and Dr. Peter Zwickl for critically reviewing our manuscript and Thomas Hrabe for aid in compiling the enormous amount of data into comprehensive tables.


  FOOTNOTES
 
Received, August 18, 2006, and in revised form, October 27, 2006.

Published, MCP Papers in Press, December 6, 2006, DOI 10.1074/mcp.M600322-MCP200

1 The abbreviations used are: VAT ATPase, valosin-containing protein-like ATPase from T. acidophilum; 2D, two-dimensional; 2DE, two-dimensional gel electrophoresis; 3D, three-dimensional; AAA ATPases, ATPases associated with a variety of cellular activities; AdoMet, S-adenosylmethionine; ED, Entner-Doudoroff; ET, electron tomography; OTCase, ornithine carbamoyltransferase; PA, polyacrylamide; SOD, superoxide dismutase. Back

2 I. Nagy, unpublished results. Back

* This work was supported by the Interaction Proteome grant, an Integrated Project funded within the Research Framework Programme 6 (FP6) of the European Commission over a period of 5 years to develop novel technologies for proteomics research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

To whom correspondence should be addressed. Tel.: 49-89-8578-2044; Fax: 49-89-8578-2641; E-mail: nagy{at}biochem.mpg.de


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