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Chaperonins are double‐ring protein folding machines fueled by ATP binding and hydrolysis. Conformational rearrangements upon ATPase cycling of the group I chaperonins, typified by the Escherichia coliGroEL/GroES system, have been thoroughly investigated by cryo‐electron microscopy and X‐ray crystallography. For archaeal group II chaperonins, however, these methods have so far failed to provide a correlation between the structural and the functional states. Here, we show that the conformation of the native αβ‐thermosome of Thermoplasma acidophilumin vitrified ice is strictly regulated by adenine nucleotides.

Valosine‐containing protein‐like ATPase from Thermoplasma acidophilumis a member of the superfamily of ATPases associated with a diversity of cellular activities and is closely related to CDC48 from yeast and p97 from higher eukaryotes and more distantly to N‐ethylmaleimide‐sensitive fusion protein. We have used electron tomography to obtain low‐resolution (2–2.5 nm) three‐dimensional maps of both the whole 500 kDa complex and the N‐terminally truncated valosine‐containing protein‐like ATPase from T. acidophilumcomplex lacking the putative substrate binding domain.

The gene encoding the α-subunit of the proteasome from Thermoplasma acidophilumwas cloned and sequenced. The gene encodes for a polypeptide with 233 amino acid and reduces a calculated molecular weight of 25870. Sequence similarity of the α-subunit with the Saccharomyces cerevislaewild-type suppressor gene sell* encoded polypeptide, which is probably identical with the subunit YC7-α of the yeast proteasome, lends support to a putative role of proteasomes in the regulation of gene expression. The significant sequence similarity to the various subunits of eukaryotic proteasomes make it likely that proteasomal proteins are encoded by one gene family of ancient origin.

The three-dimensional structure of proteasomes from the archaebacterium Thermoplasma acidophilumhas been determined to a resolution of approximately 2 nm from electron micrographs of negatively stained preparations using the method of random conical tilting. The particles turn out to be essentially cylinder-shaped barrels, 15 nm long and 11 nm wide, enclosing a tripartite inner compartiment. An account is given of some of the present limitations which prevent to attain a higher resolution and possible ways to overcome these limitations are indicated.

The subunit topography of the Thermoplasma acidophilumproteasome was determined by iminunoelectron microscopy using monospecific antibodies directed against the two constituent subunits (α,β). Anti-α-subunit IgG was found to bind to the outer disks of the cylinder- or barrel-shaped molecule, while the binding sites of the anti-β-subunit IgG were mapped on the two inner rings. Probably the homologues of the two subunits in the compositionally more complex but isomorphous eukaryotic proteasomes occupy equivalent positions.

Proteasomes reach their mature active state via a complex cascade of folding, assembly and processing events. The Rhodococcusproteasome offers a means to dissect the assembly pathway and to characterize intermediates; its four subunits (α 1, α 2, β 1, β 2) assemble efficiently in vitro with any combination of α and β. Assembly studies with wild-type and N-terminally truncated β-subunits in conjunction with refolding studies allowed to define the role of the propeptide which is two-fold: It supports the initial folding of the β-subunits and it promotes the maturation of the holoproteasomes.

Rat insulinoma 1046‐38 cells represent a model system to study β‐cell function. The mRNAs for sst1 and sst2, two of the five somatostatin receptors, were detected by reverse transcription polymerase chain reaction amplification in these cells. Displacement binding analysis suggested that sst1 represents the major somatostatin receptor subtype. The sst1 selective compound CH‐275 did not inhibit adenylyl cyclases while compounds that activated sst2 did. In contrast, CH‐275 caused a marked inhibition of voltage‐operated Ca2+channels while the sst2 specific analog octreotide elicited a less pronounced effect suggesting that in rat insulinoma 1046‐38 cells sst1 preferably mediates the inhibition of Ca2+channels.

Hitherto the biology of proteolysis in prokaryotes, particularly in archaea, is only poorly understood. We have used the tri‐peptide vinyl sulfone inhibitor carboxybenzyl‐leucyl‐leucyl‐leucine vinyl sulfone (Z‐L3VS) to study the in vivo function of proteasomes in Thermoplasma acidophilum. Z‐L3VS is a potent inhibitor of the Thermoplasmaproteasome and is capable of modifying 75 to 80% of the proteasomal β‐subunits in cell cultures. Inhibition of proteasomes has only marginal effects under normal growth conditions. Under heat shock conditions, however, the effects of proteasome inhibition are much more severe, to the extent of complete cell growth arrest. These data suggest that other proteolytic systems may exist that can compensate for the loss of proteasome function in T. acidophilum.

Based on a twelve residue master peptide comprising all five specific cleavage sites defined for the proteasome, a set of variant peptides was generated in order to probe specificity and to elucidate the mechanism which determines product size. It is shown that the rate of degradation by the 20S proteasome from Thermoplasma acidophilumdepends critically on the length of the peptide substrate. Peptides of 14 residues and longer are degraded much faster than shorter peptides although the sites of cleavage remain unchanged. The decelerated degradation of peptides shorter than 14 residues explains the accumulation of products with an average length of seven to nine residues.

© 1997 Federation of European Biochemical Societies.

The 20S proteasome, isolated from the nocardioform actinomycete Rhodococcus erythropolisstrain NI86/21, is built from two α‐type and two β‐type subunits. In order to probe the subunit topology, we have set up an expression system which allows coexpression of the genes encoding the α‐ and β‐subunits in all possible combinations. The four respective constructs obtained yielded fully assembled and proteolytically active proteasomes. Biochemical, kinetic and electron microscopy analysis allow us to rule out several of the models which were originally envisaged for the subunit topology of the Rhodococcusproteasome. The experiments further indicate that the assembly pathways of the Rhodococcusand of the Thermoplasmaproteasome differ in some important details.

Thermotoga maritimais an extremely thermophilic eubacterium and represents the deepest known branch and the most slowly evolving line within this kingdom. This bacterium possesses a sheath‐like envelope which forms characteristic balloons (the ‘toga’) around the cell poles. We have shown by electron microscopy in conjunction with image processing and by biochemical analyses that this sheath is essentially an outer membrane. Its predominant 42 kDa protein, which is regularly arrayed in vivo, has all the characteristics of the trimeric porins known from the phylogenetically very distant proteobacteria. This was an unexpected finding, which has some interesting evolutionary implications.

Proteasomes reach their mature active state via a complex cascade of folding, assembly and processing events. The Rhodococcusproteasome offers a means to dissect the assembly pathway and to characterize intermediates; its four subunits (α1, α2, β1, β2) assemble efficiently in vitro with any combination of α and β. Assembly studies with wild‐type and N‐terminally truncated β‐subunits in conjunction with refolding studies allowed to define the role of the propeptide which is two‐fold: It supports the initial folding of the β‐subunits and it promotes the maturation of the holoproteasomes.

The subunit topography of the Thermoplasma acidophilumproteasome was determined by iminunoelectron microscopy using monospecific antibodies directed against the two constituent subunits (α,β). Anti‐α‐subunit IgG was found to bind to the outer disks of the cylinder‐ or barrel‐shaped molecule, while the binding sites of the anti‐β‐subunit IgG were mapped on the two inner rings. Probably the homologues of the two subunits in the compositionally more complex but isomorphous eukaryotic proteasomes occupy equivalent positions.

The three‐dimensional structure of proteasomes from the archaebacterium Thermoplasma acidophilumhas been determined to a resolution of approximately 2 nm from electron micrographs of negatively stained preparations using the method of random conical tilting. The particles turn out to be essentially cylinder‐shaped barrels, 15 nm long and 11 nm wide, enclosing a tripartite inner compartiment. An account is given of some of the present limitations which prevent to attain a higher resolution and possible ways to overcome these limitations are indicated.

It is shown that proteasomes from the archaebacterium Thermoplasma acidophilum selectively degrade substrate proteins partially unfolded by phenylhydrazine‐ or hydrogen peroxide‐treatment. Surprisingly, the pre‐incubation of the substrate proteins with ubiquitin is also sufficient to render them susceptible to proteolytic degradation by proteasomes. We propose that, upon spontaneously associating with the substrate protein, ubiquitin exerts a chaotropic effect on it; this may involve the exposure of hydrophobic segments of the polypeptide chain which are recognized by the binding sites of the proteasome.

The thermosome, the chaperonin of the archaea, and its homologue from the cytosol of eukaryotes, known as TRiC or CCT, form a distinct subfamily of the chaperonins that does not depend on a co‐chaperonin for protein folding activity. Recent structural data obtained by cryo‐ electron microscopy and X‐ray crystallography provide the first insights into a novel mechanism remarkably different from that of the bacterial GroEL‐GroES system.

Apoptosis can be initiated in immortalized cAMP-stimulated rat ovarian granulosa cells by induction of wild-type p53 activity. Immunocytochemical studies using confocal microscopy reveal that in apoptotic, unlike in normal growing cells, the proteasomes are removed from the nucleus and accumulate within the apoptotic blebs at the periphery of the cell. In parallel, a striking reorganization of the actin cytoskeleton is observed which forms a spherical network separating the apoptotic blebs from the cytoplasmic organelles, such as mitochondria and lipid droplets which remain in the perinuclear region. The reorganization of the actin cytoskeleton as well as disappearance of proteasomes from the nucleus suggest possible function of proteasomes in apoptotic regulation.

Analysis of the degradation products from two proteins, the insulin B-chain and human hemoglobin, generated by archaebacterial Thermoplasma acidophilum20 S proteasomes, revealed an unexpectedly broad specificity. In spite of the vast number of different peptides found, they fell into a rather narrow size range. This suggests that a molecular ruler exists which determines the length of the cleavage products.

The stalks (hyphae) of a prosthecate bacterium, directly sampled from the water surface of a hot pond, show extended regular patterns on their envelope in the electron microscope. Image processing revealed a structure of the crystalline complexes which is very similar to the gross morphology of the Escherichia coliporins OmpC and OmpF. The natural two-dimensional crystal of the outer membrane protein has p3 symmetry and a lattice constant of 7.95 nm. The three-dimensional structure of the stalk porin has been determined to an almost isotropic resolution of 1.7 nm. The reconstruction revealed a complex network of channels within the membrane matrix with a triplet of pores merging into a common outlet, similar to the structure of the E. coliporin OmpF in reconstituted membranes. In addition, a blindly ending pore exists which appears to be connected to the continuous pores via small channels. The significance of the regularly arrayed porin cylinders with respect to the shape and function of the stalks is discussed.

Thermotoga maritimais an extremely thermophilic eubacterium and represents the deepest known branch and the most slowly evolving line within this kingdom. This bacterium possesses a sheath-like envelope which forms characteristic balloons (the ‘toga’) around the cell poles. We have shown by electron microscopy in conjunction with image processing and by biochemical analyses that this sheath is essentially an outer membrane. Its predominant 42 kDa protein, which is regularly arrayed in vivo, has all the characteristics of the trimeric porins known from the phylogenetically very distant proteobacteria. This was an unexpected finding, which has some interesting evolutionary implications.

Based on a twelve residue master peptide comprising all five specific cleavage sites defined for the proteasome, a set of variant peptides was generated in order to probe specificity and to elucidate the mechanism which determines product size. It is shown that the rate of degradation by the 20S proteasome from Thermoplasma acidophilumdepends critically on the length of the peptide substrate. Peptides of 14 residues and longer are degraded much faster than shorter peptides although the sites of cleavage remain unchanged. The decelerated degradation of peptides shorter than 14 residues explains the accumulation of products with an average length of seven to nine residues.

The thermosome, the chaperonin of the archaea, and its homologue from the cytosol of eukaryotes, known as TRiC or CCT, form a distinct subfamily of the chaperonins that does not depend on a co-chaperonin for protein folding activity. Recent structural data obtained by cryo- electron microscopy and X-ray crystallography provide the first insights into a novel mechanism remarkably different from that of the bacterial GroEL-GroES system.

From Methanopyrus kandleri, the most thermophilic methanogen known so far, we have purified to homogeneity a protein complex of high molecular mass. Image analysis of transmission electron micrographs revealed a barrel-shaped particle composed of two rings with 8-fold symmetry. Only one type of subunit could be detected. The corresponding gene has been cloned and sequenced. The deduced amino acid sequence shows high homology with the members of group II chaperonins. The structure of the projection and the sequence homology suggest that this particle is the first thermosome isolated from a methanogen.

Co-expression of the two genes encoding the α- and β-subunit of the Thermoplasma acidophilumthermosome in Escherichia coliyielded fully assembled hetero-oligomeric complexes (α + β). Surprisingly, also separate expression of both genes resulted in formation of hexadecameric complexes (α, β) in the bacterial cytoplasm. On electron micrographs these complexes were indistinguishable from each other and from the native thermosome. The recombinant a-complex as well as the native thermosome could be reconstituted in vitro from their dissociated subunits in the presence of Mg-ATP.

On electron micrographs, negatively stained multicatalytic proteinase molecules are viewed end-on (ring shaped) or side-on (rectangular shaped). For aurothioglucose, ammonium molybdate- and phosphotungstate-stained molecules, the dimensions measured are consistent. In contrast, uranyl acetate-staining reveals ring-shaped particles which vary in diameter between 12 and 16 nm. This is due to a partial collapse and substantial flattening of the structure. Digital image analysis of side-on views of the particles reveals a tripartite, reel-shaped structure. Within the ring-like, end-on projections of ammonium molybdate-stained molecules six local centres of mass can be discerned; their position appears to depart, however, from a true six-fold symmetry.

From the thermoacidophilic archaebacterium, Thermoplasma acidophilum, a proteolytically active particle has been isolated which is almost identical in size and shape with the multicatalytic proteinase (prosome) from rat. This result indicates that prosomes have been developed early in evolution and that they possibly serve functions common to all living cells.

Tricorn protease (TRI), a high molecular mass complex from the archaeon T. acidophilum, forms the core of a modular proteolytic system; upon interacting with low molecular mass factors intrinsic activities are enhanced and novel activities are generated. Here we characterize the first factor, F1, which turns out to be homologous with several bacterial proline iminopeptidases (PIPs). Surprisingly, it cleaves not only typical PIP substrates such as H-Pro-AMC, but a wide spectrum of amino acid substrates and several peptide substrates without a proline at the N-terminus. The pipgene encodes a 293 amino acid residue protein with a molecular mass of 33487 Da. By means of site-directed mutagenesis we identified Ser 105and His 271as the active site nucleophile and proton donor, respectively. Experiments with inactive mutant PIPs indicate that the activities elicited by interacting with TRI are contributed by PIP.

Secondary structure prediction has made great progress in recent years due to the incorporation of evolutionary information, and may be close to a point where (in combination with biochemical and low-resolution structural data) it can guide the modelling of tertiary structure in cases where no model building is possible by homology. Towards this goal it is important to gather information on the performance of prediction methods in advance of the publication of new structures. In anticipation of the soon-to-be-released structure of the 20 S proteasome from Thermoplasma acidophilum, we have applied several widely used secondary structure prediction methods to proteasome sequences and have attempted to model the putative channel in the outer proteasome rings (α-rings) based on the obtained predictions.

The decapeptide gonadotropin-releasing hormone (GnRH) is degraded by the 20 S multicatalytic proteinase complex (proteasome EC 3.4.99.46), purified from ovarian granulosa cells, at the Tyr 5Gly 6bond and to a lesser extent at the Gly 6Leu 7bond, when incubated for 2 h at 37°C. Further cleavage, at Trp 3Ser 4and Ser 4Tyr 5bonds of the neurohormone occurs only subsequently to the appearance of the initial N-terminal degradation products, (1–5)GnRH and (1–6)GnRH. Our results suggest that the sequential degradation of GnRH can serve as an important mechanism for the rapid termination of its biological activity in target cells.

The primary structure of the rat insulin-degrading enzyme (IDE) was determined by cDNA analysis. Rat IDE, as well as the previously characterized homologs from human and Drosophila, contain the carboxyl-terminal consensus sequence A/S-K-L for peroxisome targeting. A stretch of 43 bp surrounding an alternatively used polyadenylation site is highly conserved between rat and human, suggesting that it may contain important regulatory information. Northern blot analysis revealed two IDE transcripts of 3.7 and 5.5 kb in various tissues. Testis was found to be exceptional in having three different RNAs (3.7, 4.1 and 6.1 kb) at a relatively high abundance. The expression of the IDE gene in testis is correlated with sexual maturation.

The two genes encoding the constituent subunits of the Thermoplasma acidophilumproteasome were expressed in Escherichia coliyielding fully assembled molecules as shown by electron microscopy. The recombinant proteasomes were purified to homogeneity and ware shown to have proteolytic activity indistinguishable from proteasomes isolated from T. acidophilum.

The two genes encoding the constituent subunits of the Thermoplasma acidophilumproteasome were expressed in Escherichia coliyielding fully assembled molecules as shown by electron microscopy. The recombinant proteasomes were purified to homogeneity and ware shown to have proteolytic activity indistinguishable from proteasomes isolated from T. acidophilum.

The 20 S proteasome, found in eukaryotes and in the archaebacterium Thermoplasma acidophilum, forms the proteolytic core of the 26 S proteasome which is the central protease of the non‐lysosomal protein degradation pathway. Inhibitor studies have indicated that the 20 S proteasome may be an unusual type of cysteine or serine protease and a recent study of the Thermoplasmaβ subunit has indicated that it carries the proteolytic activity. We have attempted to obtain information on the nature of the active site by mutating the only cysteine, both histidines and two completely conserved aspartates in the archaebacterial complex as well as all serines of the β subunit, without decreasing the catalytic activity of the enzyme to any significant extent. Indeed, mutation of the conserved aspartate in the β subunit increased the activity of the proteasome threefold. We conclude that the proteasome is not a cysteine or serine protease.

A large protein complex (approx. 2000 kDa) was found in the cytosol of the hyperthermophilic archaebacterium Staphylothermus marinus. The purified protein was shown to be a homomultimer of 93 kDa subunits, the primary structure of which was determined by nucleotide sequence analysis. The protein belongs to the family of phosphoenolpyruvate‐utilizing enzymes and represents the first member characterized in archaebacteria. Its homomultimeric organisation differs from the typically dimeric structure of its eubacterial and eukaryotic counterparts.

The gene encoding the α‐subunit of the proteasome from Thermoplasma acidophilumwas cloned and sequenced. The gene encodes for a polypeptide with 233 amino acid and reduces a calculated molecular weight of 25870. Sequence similarity of the α‐subunit with the Saccharomyces cerevislaewild‐type suppressor gene sell* encoded polypeptide, which is probably identical with the subunit YC7‐α of the yeast proteasome, lends support to a putative role of proteasomes in the regulation of gene expression. The significant sequence similarity to the various subunits of eukaryotic proteasomes make it likely that proteasomal proteins are encoded by one gene family of ancient origin.

The stalks (hyphae) of a prosthecate bacterium, directly sampled from the water surface of a hot pond, show extended regular patterns on their envelope in the electron microscope. Image processing revealed a structure of the crystalline complexes which is very similar to the gross morphology of the Escherichia coliporins OmpC and OmpF. The natural two‐dimensional crystal of the outer membrane protein has p3 symmetry and a lattice constant of 7.95 nm. The three‐dimensional structure of the stalk porin has been determined to an almost isotropic resolution of 1.7 nm. The reconstruction revealed a complex network of channels within the membrane matrix with a triplet of pores merging into a common outlet, similar to the structure of the E. coliporin OmpF in reconstituted membranes. In addition, a blindly ending pore exists which appears to be connected to the continuous pores via small channels. The significance of the regularly arrayed porin cylinders with respect to the shape and function of the stalks is discussed.

From Methanopyrus kandleri, the most thermophilic methanogen known so far, we have purified to homogeneity a protein complex of high molecular mass. Image analysis of transmission electron micrographs revealed a barrel‐shaped particle composed of two rings with 8‐fold symmetry. Only one type of subunit could be detected. The corresponding gene has been cloned and sequenced. The deduced amino acid sequence shows high homology with the members of group II chaperonins. The structure of the projection and the sequence homology suggest that this particle is the first thermosome isolated from a methanogen.

The 20 S proteasome, found in eukaryotes and in the archaebacterium Thermoplasma acidophilum, forms the proteolytic core of the 26 S proteasome which is the central protease of the non-lysosomal protein degradation pathway. Inhibitor studies have indicated that the 20 S proteasome may be an unusual type of cysteine or serine protease and a recent study of the Thermoplasmaβ subunit has indicated that it carries the proteolytic activity. We have attempted to obtain information on the nature of the active site by mutating the only cysteine, both histidines and two completely conserved aspartates in the archaebacterial complex as well as all serines of the β subunit, without decreasing the catalytic activity of the enzyme to any significant extent. Indeed, mutation of the conserved aspartate in the β subunit increased the activity of the proteasome threefold. We conclude that the proteasome is not a cysteine or serine protease.

A large protein complex (approx. 2000 kDa) was found in the cytosol of the hyperthermophilic archaebacterium Staphylothermus marinus. The purified protein was shown to be a homomultimer of 93 kDa subunits, the primary structure of which was determined by nucleotide sequence analysis. The protein belongs to the family of phosphoenolpyruvate-utilizing enzymes and represents the first member characterized in archaebacteria. Its homomultimeric organisation differs from the typically dimeric structure of its eubacterial and eukaryotic counterparts.

It is shown that proteasomes from the archaebacterium Thermoplasma acidophilum selectively degrade substrate proteins partially unfolded by phenylhydrazine- or hydrogen peroxide-treatment. Surprisingly, the pre-incubation of the substrate proteins with ubiquitin is also sufficient to render them susceptible to proteolytic degradation by proteasomes. We propose that, upon spontaneously associating with the substrate protein, ubiquitin exerts a chaotropic effect on it; this may involve the exposure of hydrophobic segments of the polypeptide chain which are recognized by the binding sites of the proteasome.

From the thermoacidophilic archaebacterium, Thermoplasma acidophilum, a proteolytically active particle has been isolated which is almost identical in size and shape with the multicatalytic proteinase (prosome) from rat. This result indicates that prosomes have been developed early in evolution and that they possibly serve functions common to all living cells.

Analysis of the degradation products from two proteins, the insulin B‐chain and human hemoglobin, generated by archaebacterial Thermoplasma acidophilum20 S proteasomes, revealed an unexpectedly broad specificity. In spite of the vast number of different peptides found, they fell into a rather narrow size range. This suggests that a molecular ruler exists which determines the length of the cleavage products.

Tricorn protease (TRI), a high molecular mass complex from the archaeon T. acidophilum, forms the core of a modular proteolytic system; upon interacting with low molecular mass factors intrinsic activities are enhanced and novel activities are generated. Here we characterize the first factor, F1, which turns out to be homologous with several bacterial proline iminopeptidases (PIPs). Surprisingly, it cleaves not only typical PIP substrates such as H‐Pro‐AMC, but a wide spectrum of amino acid substrates and several peptide substrates without a proline at the N‐terminus. The pipgene encodes a 293 amino acid residue protein with a molecular mass of 33487 Da. By means of site‐directed mutagenesis we identified Ser105and His271as the active site nucleophile and proton donor, respectively. Experiments with inactive mutant PIPs indicate that the activities elicited by interacting with TRI are contributed by PIP.

Apoptosis can be initiated in immortalized cAMP‐stimulated rat ovarian granulosa cells by induction of wild‐type p53 activity. Immunocytochemical studies using confocal microscopy reveal that in apoptotic, unlike in normal growing cells, the proteasomes are removed from the nucleus and accumulate within the apoptotic blebs at the periphery of the cell. In parallel, a striking reorganization of the actin cytoskeleton is observed which forms a spherical network separating the apoptotic blebs from the cytoplasmic organelles, such as mitochondria and lipid droplets which remain in the perinuclear region. The reorganization of the actin cytoskeleton as well as disappearance of proteasomes from the nucleus suggest possible function of proteasomes in apoptotic regulation.

Secondary structure prediction has made great progress in recent years due to the incorporation of evolutionary information, and may be close to a point where (in combination with biochemical and low‐resolution structural data) it can guide the modelling of tertiary structure in cases where no model building is possible by homology. Towards this goal it is important to gather information on the performance of prediction methods in advance of the publication of new structures. In anticipation of the soon‐to‐be‐released structure of the 20 S proteasome from Thermoplasma acidophilum, we have applied several widely used secondary structure prediction methods to proteasome sequences and have attempted to model the putative channel in the outer proteasome rings (α‐rings) based on the obtained predictions.

TF55‐like factor from Sulfolobus solfataricuswas purified to homogeneity and analyzed by electron microscopy and image analysis to determine the symmetries of these particles. Three different procedures were used to analyze the electron micrographs: (1) fuzzy‐set based classification of the particles according to their rotational power spectra; (2) multivariate statistical analysis based on singular value decomposition; (3) circular harmonic analysis. Averages obtained from the three methods show unequivocally that the TF55‐like complex presents a 9‐fold symmetry.