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W2120340031 abstract "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. 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 (1Schleper C. Puehler G. Holz I. Gambacorta A. Janekovic D. Santarius U. Klenk H.P. Zillig W. Picrophilus gen-nov, fam-nov—a novel aerobic, heterotrophic, thermoacidophilic genus and family comprising archaea capable of growth around pH-0..J. Bacteriol. 1995; 177: 7050-7059Crossref PubMed Scopus (212) Google Scholar). 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 (2Darland G. Brock T.D. Samsonof W. Conti S.F. Thermophilic, acidophilic mycoplasma isolated from a coal refuse pile..Science. 1970; 170: 1416-1418Crossref PubMed Scopus (233) Google Scholar). 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 (3Smith P.F. Langwort T.A. Mayberry W.R. Hougland A.E. Characterization of membranes of Thermoplasma acidophilum..J. Bacteriol. 1973; 116: 1019-1028Crossref PubMed Google Scholar). 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 (4Ruepp A. Graml W. Santos-Martinez M.L. Koretle K.K. Volker C. Mewes H.W. Frishman D. Stocker S. Lupas A.N. Baumeister W. The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum..Nature. 2000; 407: 508-513Crossref PubMed Scopus (341) Google Scholar). Although many macromolecular assemblies of T. acidophilum like the proteasome, thermosome, valosin-containing protein-like ATPase from T. acidophilum (VAT ATPase), 1The 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. 1The 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. 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” (5Nickell S. Kofler C. Leis A.P. Baumeister W. A visual approach to proteomics..Nat. Rev. Mol. Cell. Biol. 2006; 7: 225-230Crossref PubMed Scopus (182) Google Scholar) 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 (6Frangakis A.S. Bohm J. Forster F. Nickell S. Nicastro D. Typke D. Hegerl R. Baumeister W. Identification of macromolecular complexes in cryoelectron tomograms of phantom cells..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14153-14158Crossref PubMed Scopus (204) Google Scholar). 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.DISCUSSIONThe 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 (47Ram R.J. VerBerkmoes N.C. Thelen M.P. Tyson G.W. Baker B.J. Blake R.C. Shah M. Hettich R.L. Banfield J.F. Community proteomics of a natural microbial biofilm..Science. 2005; 308: 1915-1920Crossref PubMed Google Scholar) 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 (48Davies M.J. The oxidative environment and protein damage..Biochim. Biophys. Acta. 2005; 1703: 93-109Crossref PubMed Scopus (1073) Google Scholar). 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 (35Larsson K.M. Jordan A. Eliasson R. Reichard P. Logan D.T. Nordlund P. Structural mechanism of allosteric substrate specificity regulation in a ribonucleotide reductase..Nat. Struct. Mol. Biol. 2004; 11: 1142-1149Crossref PubMed Scopus (74) Google Scholar), and we found 10 proteins of the B12 biosynthesis pathway indicating that most probably this vitamin is produced de novo.Koonin et al. (23Koonin E.V. Wolf Y.I. Aravind L. Prediction of the archaeal exosome and its connections with the proteasome and the translation and transcription machineries by a comparative-genomic approach..Genome Res. 2001; 11: 240-252Crossref PubMed Scopus (198) Google Scholar) 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 (49Evguenieva-Hackenberg E. Walter P. Hochleitner E. Lottspeich F. Klug G. An exosome-like complex in Sulfolobus solfataricus..EMBO Rep. 2003; 4: 889-893Crossref PubMed Scopus (112) Google Scholar). 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 β-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 (50Price G.D. Badger M.R. Evidence for the role of carboxysomes in the cyanobacterial CO2-concentrating mechanism..Can. J. Bot.-Rev. Can. Bot. 1991; 69: 963-973Crossref Google Scholar, 51Orus M.I. Rodriguez-Buey M.L. Marco E. Fernandez-Valiente E. Changes in carboxysome structure and grouping and in photosynthetic affinity for inorganic carbon in Anabaena strain PCC 7119 (cyanophyta) in response to modification of CO2 and Na+ supply..Plant Cell Physiol. 2001; 42: 46-53Crossref PubMed Scopus (12) Google Scholar); they have a size of 120 nm and serve to protect ribulose-1,5-bisphosphate carboxylase/oxygenase (52Shively J.M. van Keulen G. Meijer W.G. Something from almost nothing: carbon dioxide fixation in chemoautotrophs..Annu. Rev. Microbiol. 1998; 52: 191-230Crossref PubMed Scopus (224) Google Scholar). 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 (53Wanson J.C. Drochman P. Rabbit skeletal muscle glycogen—a morphological and biochemical study of glycogen β-particles isolated by precipitation-centrifugation method..J. Cell Biol. 1968; 38: 130-150Crossref PubMed Scopus (111) Google Scholar). The electron microscopy analysis of glycogen is available (53Wanson J.C. Drochman P. Rabbit skeletal muscle glycogen—a morphological and biochemical study of glycogen β-particles isolated by precipitation-centrifugation method..J. Cell Biol. 1968; 38: 130-150Crossref PubMed Scopus (111) Google Scholar), 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. The archaeon Thermoplasma acidophilum is a member of the Euryarchaeota lineage of the Archaea (1Schleper C. Puehler G. Holz I. Gambacorta A. Janekovic D. Santarius U. Klenk H.P. Zillig W. Picrophilus gen-nov, fam-nov—a novel aerobic, heterotrophic, thermoacidophilic genus and family comprising archaea capable of growth around pH-0..J. Bacteriol. 1995; 177: 7050-7059Crossref PubMed Scopus (212) Google Scholar). 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 (2Darland G. Brock T.D. Samsonof W. Conti S.F. Thermophilic, acidophilic mycoplasma isolated from a coal refuse pile..Science. 1970; 170: 1416-1418Crossref PubMed Scopus (233) Google Scholar). 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 (3Smith P.F. Langwort T.A. Mayberry W.R. Hougland A.E. Characterization of membranes of Thermoplasma acidophilum..J. Bacteriol. 1973; 116: 1019-1028Crossref PubMed Google Scholar). 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 (4Ruepp A. Graml W. Santos-Martinez M.L. Koretle K.K. Volker C. Mewes H.W. Frishman D. Stocker S. Lupas A.N. Baumeister W. The genome sequence of the thermoacidophilic scavenger Thermoplasma acidophilum..Nature. 2000; 407: 508-513Crossref PubMed Scopus (341) Google Scholar). Although many macromolecular assemblies of T. acidophilum like the proteasome, thermosome, valosin-containing protein-like ATPase from T. acidophilum (VAT ATPase), 1The 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. 1The 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. 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” (5Nickell S. Kofler C. Leis A.P. Baumeister W. A visual approach to proteomics..Nat. Rev. Mol. Cell. Biol. 2006; 7: 225-230Crossref PubMed Scopus (182) Google Scholar) 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 (6Frangakis A.S. Bohm J. Forster F. Nickell S. Nicastro D. Typke D. Hegerl R. Baumeister W. Identification of macromolecular complexes in cryoelectron tomograms of phantom cells..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14153-14158Crossref PubMed Scopus (204) Google Scholar). 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. DISCUSSIONThe 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 (47Ram R.J. VerBerkmoes N.C. Thelen M.P. Tyson G.W. Baker B.J. Blake R.C. Shah M. Hettich R.L. Banfield J.F. Community proteomics of a natural microbial biofilm..Science. 2005; 308: 1915-1920Crossref PubMed Google Scholar) 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 (48Davies M.J. The oxidative environment and protein damage..Biochim. Biophys. Acta. 2005; 1703: 93-109Crossref PubMed Scopus (1073) Google Scholar). 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 (35Larsson K.M. Jordan A. Eliasson R. Reichard P. Logan D.T. Nordlund P. Structural mechanism of allosteric substrate specificity regulation in a ribonucleotide reductase..Nat. Struct. Mol. Biol. 2004; 11: 1142-1149Crossref PubMed Scopus (74) Google Scholar), and we found 10 proteins of the B12 biosynthesis pathway indicating that most probably this vitamin is produced de novo.Koonin et al. (23Koonin E.V. Wolf Y.I. Aravind L. Prediction of the archaeal exosome and its connections with the proteasome and the translation and transcription machineries by a comparative-genomic approach..Genome Res. 2001; 11: 240-252Crossref PubMed Scopus (198) Google Scholar) 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 (49Evguenieva-Hackenberg E. Walter P. Hochleitner E. Lottspeich F. Klug G. An exosome-like complex in Sulfolobus solfataricus..EMBO Rep. 2003; 4: 889-893Crossref PubMed Scopus (112) Google Scholar). 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 β-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 (50Price G.D. Badger M.R. Evidence for the role of carboxysomes in the cyanobacterial CO2-concentrating mechanism..Can. J. Bot.-Rev. Can. Bot. 1991; 69: 963-973Crossref Google Scholar, 51Orus M.I. Rodriguez-Buey M.L. Marco E. Fernandez-Valiente E. Changes in carboxysome structure and grouping and in photosynthetic affinity for inorganic carbon in Anabaena strain PCC 7119 (cyanophyta) in response to modification of CO2 and Na+ supply..Plant Cell Physiol. 2001; 42: 46-53Crossref PubMed Scopus (12) Google Scholar); they have a size of 120 nm and serve to protect ribulose-1,5-bisphosphate carboxylase/oxygenase (52Shively J.M. van Keulen G. Meijer W.G. Something from almost nothing: carbon dioxide fixation in chemoautotrophs..Annu. Rev. Microbiol. 1998; 52: 191-230Crossref PubMed Scopus (224) Google Scholar). 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 (53Wanson J.C. Drochman P. Rabbit skeletal muscle glycogen—a morphological and biochemical study of glycogen β-particles isolated by precipitation-centrifugation method..J. Cell Biol. 1968; 38: 130-150Crossref PubMed Scopus (111) Google Scholar). The electron microscopy analysis of glycogen is available (53Wanson J.C. Drochman P. Rabbit skeletal muscle glycogen—a morphological and biochemical study of glycogen β-particles isolated by precipitation-centrifugation method..J. Cell Biol. 1968; 38: 130-150Crossref PubMed Scopus (111) Google Scholar), 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. 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 (47Ram R.J. VerBerkmoes N.C. Thelen M.P. Tyson G.W. Baker B.J. Blake R.C. Shah M. Hettich R.L. Banfield J.F. Community proteomics of a natural microbial biofilm..Science. 2005; 308: 1915-1920Crossref PubMed Google Scholar) 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 (48Davies M.J. The oxidative environment and protein damage..Biochim. Biophys. Acta. 2005; 1703: 93-109Crossref PubMed Scopus (1073) Google Scholar). 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 (35Larsson K.M. Jordan A. Eliasson R. Reichard P. Logan D.T. Nordlund P. Structural mechanism of allosteric substrate specificity regulation in a ribonucleotide reductase..Nat. Struct. Mol. Biol. 2004; 11: 1142-1149Crossref PubMed Scopus (74) Google Scholar), and we found 10 proteins of the B12 biosynthesis pathway indicating that most probably this vitamin is produced de novo. Koonin et al. (23Koonin E.V. Wolf Y.I. Aravind L. Prediction of the archaeal exosome and its connections with the proteasome and the translation and transcription machineries by a comparative-genomic approach..Genome Res. 2001; 11: 240-252Crossref PubMed Scopus (198) Google Scholar) 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 (49Evguenieva-Hackenberg E. Walter P. Hochleitner E. Lottspeich F. Klug G. An exosome-like complex in Sulfolobus solfataricus..EMBO Rep. 2003; 4: 889-893Crossref PubMed Scopus (112) Google Scholar). 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 β-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 (50Price G.D. Badger M.R. Evidence for the role of carboxysomes in the cyanobacterial CO2-concentrating mechanism..Can. J. Bot.-Rev. Can. Bot. 1991; 69: 963-973Crossref Google Scholar, 51Orus M.I. Rodriguez-Buey M.L. Marco E. Fernandez-Valiente E. Changes in carboxysome structure and grouping and in photosynthetic affinity for inorganic carbon in Anabaena strain PCC 7119 (cyanophyta) in response to modification of CO2 and Na+ supply..Plant Cell Physiol. 2001; 42: 46-53Crossref PubMed Scopus (12) Google Scholar); they have a size of 120 nm and serve to protect ribulose-1,5-bisphosphate carboxylase/oxygenase (52Shively J.M. van Keulen G. Meijer W.G. Something from almost nothing: carbon dioxide fixation in chemoautotrophs..Annu. Rev. Microbiol. 1998; 52: 191-230Crossref PubMed Scopus (224) Google Scholar). 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 (53Wanson J.C. Drochman P. Rabbit skeletal muscle glycogen—a morphological and biochemical study of glycogen β-particles isolated by precipitation-centrifugation method..J. Cell Biol. 1968; 38: 130-150Crossref PubMed Scopus (111) Google Scholar). The electron microscopy analysis of glycogen is available (53Wanson J.C. Drochman P. Rabbit skeletal muscle glycogen—a morphological and biochemical study of glycogen β-particles isolated by precipitation-centrifugation method..J. Cell Biol. 1968; 38: 130-150Crossref PubMed Scopus (111) Google Scholar), 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. 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." @default.
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W2120340031 date "2007-03-01" @default.
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W2120340031 title "Proteomics Analysis of Thermoplasma acidophilum with a Focus on Protein Complexes" @default.
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W2120340031 doi "https://doi.org/10.1074/mcp.m600322-mcp200" @default.
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