The methanogenic archaeon Methanosarcina thermophila TM-1 possesses a high-affinity glycine betaine transporter involved in osmotic adaptation.

Methanogenic Archaea are found in a wide range of environments and use several strategies to adjust to changes in extracellular solute concentrations. One methanogenic archaeon, Methanosarcina thermophila TM-1, can adapt to various osmotic conditions by synthesis of alpha-glutamate and a newly discovered compatible solute, Ne-acetyl-beta-lysine, or by accumulation of glycine betaine (betaine) and potassium ions from the environment. Since betaine transport has not been characterized for any of the methanogenic Archaea, we examined the uptake of this solute by M. thermophila TM-1. When cells were grown in mineral salts media containing from 0.1 to 0.8 M NaC1, M. thermophila accumulated betaine in concentrations up to 140 times those of a concentration gradient within 10 min of exposure to the solute. The betaine uptake system consisted of a single, high-affinity transporter with an apparent K3 of 10 microM and an apparent maximum transport velocity of 1.15 nmol/min/mg of protein. The transporter appeared to be specific for betaine, since potential substrates, including glycine, sarcosine, dimethyl glycine, choline, and proline, did not significantly inhibit betaine uptake. M. thermophila TM-1 cells can also regulate the capacity for betaine accumulation, since the rate of betaine transport was reduced in cells pregrown in a high-osmolarity medium when 500 microM betaine was present. Betaine transport appears to be H+ and/or Na+ driven, since betaine transport was inhibited by several types of protonophores and sodium ionophores.     

Gene cloning and expression and characterization of a toxin-sensitive protein phosphatase from the methanogenic archaeon Methanosarcina thermophila TM-1.

With oligonucleotides modelled after conserved regions within the protein-serine/threonine phosphatases (PPs) of the PP1/2A/2B superfamily, the gene for the archaeal protein phosphatase PP1-arch2 was identified, cloned, and sequenced from the methanogenic archaeon Methanosarcina thermophila TM-1. The DNA-derived amino acid sequence of PP1-arch2 exhibited a high degree of sequence identity, 27 to 31%, with members of the PP1/2A/2B superfamily such as PP1-arch1 from Sulfolobus solfataricus, PP1alpha from rats, PP2A from Saccharomyces cerevisiae, and PP2B from humans. The activity of the recombinant PP1-arch2 was sensitive to several naturally occurring microbial toxins known to potently inhibit eucaryal PP1 and PP2A, including microcystin-LR, okadaic acid, tautomycin, and calyculin A.     

Polysaccharide reserve material in the acetotrophic methanogen,Methanosarcina thermophila strain TM-1: accumulation and mobilization

The ability of Methanosarcina thermophila strain TM-1 to store a reserve polysaccharide was studied using both biochemical methods and thin-section electron microscopy. When grown under conditions of excess carbon and energy (either methanol or acetate) and limiting nitrogen, M. thermophila accumulated a polysaccharide which could be hydrolyzed to glucose by the enzyme amyloglucosidase. This polysaccharide reached levels of 20 mg polysaccharide per g protein in nitrogen-limited cells, while cells limited for carbon, as well as cells in the exponential phase of growth, did not accumulate significant amounts of this polysaccharide. Thin-section electron micrographs of M. thermophila showed glycogen-like inclusion granules in nitrogen-limited cells but not in carbon-limited or exponential-phase cells. These granules were stained by a polysaccharide-specific staining procedure, the PATO stain. The polysaccharide was purified from cell extracts, the iodine-polysaccharide complex gave a maximum absorption at between 500 and 510 nm. The polysaccharide was mobilized within 21 h by cells starved for a carbon/energy source. N-Limited (polysaccharide-containing) acetategrown cells could shift to methanogenesis from methanol more quickly than did C-limited acetate-grown cells lacking polysaccharide, and ATP levels remained higher in N-limited cells. The results are consistant with the hypothesis that this polysaccharide can provide carbon and energy for metabolic shifts but other storage compounds, such as polyphosphate, may also play a similar role.     

The assembly and intermolecular properties of the hsp70-Hop-hsp90 molecular chaperone complex

The highly coordinated interactions of several molecular chaperones, including hsp70 and hsp90, are required for the folding and conformational regulation of a variety of proteins in eukaryotic cells, such as steroid hormone receptors and many other signal transduction regulators. The protein called Hop serves as an adaptor protein for hsp70 and hsp90 and is thought to optimize their functional cooperation. Here we characterize the assembly of the hsp70-Hop-hsp90 complex and reveal interactions that cause conformational changes between the proteins in the complex. We found that hsp40 plays an integral role in the assembly by enhancing the binding of hsp70 to the Hop complex. This is accomplished by stimulating the conversion of hsp70-ATP to hsp70-ADP, the hsp70 conformation favored for Hop binding. The hsp70-Hop-hsp90 complex is highly dynamic, as has been observed previously for hsp90 in its interaction with client proteins. Nonetheless, hsp90 binds with high affinity to Hop (K(d) = 90 nm), and this binding is not affected by hsp70. hsp70 binds with lower affinity to Hop (K(d) = 1.3 microm) on its own, but this affinity is increased (K(d) = 250 nm) in the presence of hsp90. hsp90 also reduces the number of hsp70 binding sites on the Hop dimer from two sites in the absence of hsp90 to one site in its presence. Hop can inhibit the ATP binding and p23 binding activity of hsp90, yet this can be reversed if hsp70 is present in the complex. Taken together, our results suggest that the assembly of hsp70-Hop-hsp90 complexes is selective and influences the conformational state of each protein.     

Downstream caspases are novel targets for the antiapoptotic activity of the molecular chaperone hsp70

The response of cancer cells to apoptosis-inducing agents can be characterized by 2 opposing factors, the proapoptotic caspase cascade and the antiapoptotic stress protein Hsp70. We show here that these factors interact in U-937 leukemia cells induced to apoptosis with anticancer drugs, etoposide and adriamycin (ADR). The protective effect of Hsp70 was verified using 2 approaches: mild heat stress and transfection-mediated overexpression of the Hsp70 gene. The increase in Hsp70 levels attained by these 2 methods was found to postpone caspase activation for 12-18 hours. An in vitro assay was developed using mouse myeloma NS0/1 cells, which lack the expression of Hsp70. Measurement of DEVD-ase activity in extracts of apoptotic NS0/1 cells incubated with purified Hsp70 showed that Hsp70 reduced caspase activity by up to 50% of its control value in a dose-dependent manner. The hypothesis that the inhibitory effect of Hsp70 on caspase-3/7 activity related to a direct interaction between Hsp70 and the caspases was tested by reciprocal immunoprecipitations and Far-western analyses. These tests were performed with extracts of Hsp70-overexpressing, control, and ADR-treated U-937 cells and using anti-caspase-3, caspase-7, and anti-Hsp70 antibodies, and the data clearly showed that Hsp70 was able to interact with the proforms of these caspases in cell lysates and with reconstituted purified proteins but did not bind the activated forms of either caspase-3 or -7. This association was also corroborated by a novel, enzyme-linked immunosorbent assay-like assay, protein interaction assay, that combined the advantages of immunoprecipitation and immunoblotting in a 96-well microplate-based assay. Thus, Hsp70 may act to suppress caspase-dependent apoptotic signaling through binding the precursor forms of both caspase-3 and caspase-7 and preventing their maturation.     

The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.     

Structure, organization, and expression of genes coding for envelope components in the archaeon Methanosarcina mazei S-6

The antigenic mosaics of archaeal species are complex and lead to the distinction of different immunotypes. We began the dissection of the antigenic mosaic of the methanogen Methanosarcina mazei S-6 by gene cloning and sequencing. The analysis of the sequence, organization, and in vitro (heterologous) and in vivo expression of two three-gene clusters that encode proteins localized to the cell envelope and that are recognized by antibodies for surface structures is presented in this report. The amino acid sequences and compositions share characteristics with S-layer proteins and, most notably, have repeats of conserved sequences and secondary structures. Expressed genes produced proteins with a tendency to oligomerize, and one of these proteins was susceptible to breakdown at regular intervals. Altogether, the data reveal a modular system (clusters of homologous genes, proteins of similar sequences with conserved repeats) seemingly suitable for assembling an enormous variety of final molecular structures by rearranging and combining genes, proteins, and repeats, and thus generate the observed wide spectrum of antigenic diversity. Received: 26 June 1997 / Accepted: 5 November 1997     

The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding.

The properties of molecular chaperones in protein-assisted refolding were examined in vitro using recombinant human cytosolic chaperones hsp90, hsc70, hsp70 and hdj-1, and unfolded beta-galactosidase as the substrate. In the presence of hsp70 (hsc70), hdj-1 and either ATP or ADP, denatured beta-galactosidase refolds and forms enzymatically active tetramers. Interactions between hsp90 and non-native beta-galactosidase neither lead to refolding nor stimulate hsp70- and hdj-1-dependent refolding. However, hsp90 in the absence of nucleotide can maintain the non-native substrate in a 'folding-competent' state which, upon addition of hsp70, hdj-1 and nucleotide, leads to refolding. The refolding activity of hsp70 and hdj-1 is effective across a broad range of temperatures from 22 degrees C to 41 degrees C, yet at extremely low (4 degrees C) or high (>41 degrees C) temperatures refolding activity is reversibly inhibited. These results reveal two distinct features of chaperone activity in which a non-native substrate can be either maintained in a stable folding-competent state or refolded directly to the native state; first, that the refolding activity itself is temperature sensitive and second, that hsp90, hsp70 (hsc70) and hdj-1 each have distinct roles in these processes.     

Nucleotide exchange factor for the yeast Hsp70 molecular chaperone Ssa1p

We report on the identification of Fes1p (yBR101cp) as a cytosolic homologue of Sls1p, an endoplasmic reticulum (ER) protein previously shown to act as a nucleotide exchange factor for yeast BiP (M. Kabani, J.-M. Beckerich, and C. Gaillardin, Mol. Cell. Biol. 20:6923-6934, 2000). We found that Fes1p associates preferentially to the ADP-bound form of the cytosolic Hsp70 molecular chaperone Ssa1p and promotes nucleotide release. Fes1p activity was shown to be compartment and species specific since Sls1p and Escherichia coli GrpE could not substitute for Fes1p. Surprisingly, whereas Sls1p stimulated the ATPase activity of BiP in cooperation with luminal J proteins, Fes1p was shown to inhibit the Ydj1p-mediated activation of Ssa1p ATPase activity in steady-state and single-turnover assays. Disruption of FES1 in several wild-type backgrounds conferred a strong thermosensitive phenotype but partially rescued ydj1-151 thermosensitivity. The Delta fes1 strain was proficient for posttranslational protein translocation, as well as for the ER-associated degradation of two substrates. However, the Delta fes1 mutant showed increased cycloheximide sensitivity and a general translational defect, suggesting that Fes1p acts during protein translation, a process in which Ssa1p and Ydj1p are known to be involved. In support of this hypothesis, Fes1p was found to be associated with ribosomes.     

The induction mechanism of the molecular chaperone HSP70 in the gastric mucosa by Geranylgeranylacetone (HSP-inducer)

To elucidate the induction mechanism of HSP70 by geranylgeranylacetone (GGA), we investigated GGA specific binding proteins using a GGA-affinity column. Alteration of chaperone activity of HSP70 and binding affinity of HSP70 to heat shock factor-1 (HSF-1) was evaluated in the presence or absence of GGA. The binding domain of HSP70 to GGA was also analyzed. A 70-kDa protein eluted by 10 mM GGA from the GGA-affinity column was identical to constitutively expressed HSP70 on immunoblotting. GGA-binding domain of HSP70 was C-terminal of the protein as peptide-binding domain (HSP70C). The chaperone activity of HSP70 and recombinant HSP70C was suppressed by GGA. Furthermore, dissociation of the HSP70 from HSF-1 was observed in the presence of GGA. GGA preferentially binds to the C-terminal of HSP70 which binds to HSF-1. After dissociation of HSP70, free HSF-1 could acquire the ability to bind to HSE (the promoter region of HSP70) gene.     

Evolution of a Protein-Folding Machine: Genomic and Evolutionary Analyses Reveal Three Lineages of the Archaeal hsp70(dnaK) Gene

The stress chaperone protein Hsp70 (DnaK) (abbreviated DnaK) and its co-chaperones Hsp40(DnaJ) (or DnaJ) and GrpE are universal in bacteria and eukaryotes but occur only in some archaea clustered in the order 5′-grpE-dnaK-dnaJ-3′ in a locus termed Locus I. Three structural varieties of Locus I, termed Types I, II, and III, were identified, respectively, in Methanosarcinales, in Thermoplasmatales and Methanothermobacter thermoautotrophicus, and in Halobacteriales. These Locus I types corresponded to three groups identified by phylogenetic trees of archaeal DnaK proteins including the same archaeal subdivisions. These archaeal DnaK groups were not significantly interrelated, clustering instead with DnaKs from three bacterial lineages, Methanosarcinales with Firmicutes, Thermoplasmatales and M. thermoautotrophicus with Thermotoga, and Halobacteriales with Actinobacteria, suggesting that the three archaeal types of Locus I were acquired by independent events of lateral gene transfer. These associations, however, lacked strong bootstrap support and were sensitive to dataset choice and tree-reconstruction method. Structural features of dnaK loci in bacteria revealed that Methanosarcinales and Firmicutes shared a similar structure, also common to most other bacterial groups. Structural differences were observed instead in Thermotoga compared to Thermoplasmatales and M. thermoautotrophicus, and in Actinobacteria compared to Halobacteriales. It was also found that the association between the DnaK sequences from Halobacteriales and Actinobacteria likely reflects common biases in their amino acid compositions. Although the loci structural features and the DnaK trees suggested the possibility of lateral gene transfer between Firmicutes and Methanosarcinales, the similarity between the archaeal and the ancestral bacterial loci favors the more parsimonious hypothesis that all archaeal sequences originated from a unique prokaryotic ancestor. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Stephen Freeland]     

The molecular chaperone hsp70 interacts with the cytosolic II-III loop of the Cav2.3 E-type voltage-gated Ca2+ channel.

Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coexist in excitable cells and participate in synaptic differentiation, secretion, transmitter release, and neuronal plasticity. Ca2+ ions entering cells trigger these events through their interaction with the ion channel itself or through Ca2+ binding to target proteins initiating signalling cascades at cytosolic loops of the ion conducting subunit (Cava1). These loops interact with target proteins in a Ca2+-dependent or independent manner. In Cav2.3-containing channels the cytosolic linker between domains II and III confers a novel Ca2+ sensitivity to E-type Ca2+ channels including phorbol ester sensitive signalling via protein kinase C (PKC) in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3 Ca2+ channels, protein interaction partners of the II-III loop were identified. FLAG-tagged II-III - loop of human Cav2.3 was over-expressed in HEK 293 cells, and the molecular chaperone hsp70, which is known to interact with PKC, was identified as a novel functional interaction partner. Immunopurified II-III loop-protein of neuronal and endocrine Cav2.3 splice variants stimulate autophosphorylation of PKCa, leading to the suggestion that hsp70--binding to the II-III loop--may act as an adaptor for Ca2+ dependent targeting of PKC to E-type Ca2+ channels.     

Molecular identification and expression of heat shock cognate 70 (hsc70) and heat shock protein 70 (hsp70) genes in the Pacific oyster Crassostrea gigas

The 70-kDa heat shock protein (Hsp) family is composed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) family members. We sequenced 2 genes encoding an Hsp70 and an Hsc70 in the Pacific oyster Crassostrea gigas. The Cghsc70 gene contained introns, whereas the Cghsp70 gene did not. Moreover, the corresponding amino acid sequences of the 2 genes presented all the characteristic motifs of the Hsp70 family. We also investigated the expression of Hsp70 in tissues of oysters experimentally exposed to metal. A recombinant Hsc72 was used as an antigen to produce a polyclonal antibody to quantify soluble Hsp70 by enzyme-linked immunosorbent assay in protein samples extracted from oysters. Our results showed that metals (copper and cadmium) induced a decrease in cytosolic Hsp70 level in gills and digestive gland of oysters experimentally exposed to metal. These data suggest that metals may inhibit stress protein synthesis.     

Cloning of the hsp70 (dnaK) genes from Rhizobium meliloti and Pseudomonas cepacia: phylogenetic analyses of mitochondrial origin based on a highly conserved protein sequence.

The genes for hsp70 (or dnaK) have been cloned and sequenced from Rhizobium meliloti and Pseudomonas cepacia, two bacterial species belonging to the alpha- and beta-subdivisions of gram-negative proteobacteria, respectively. On the basis of global alignment of HSP70 proteins, several sequence signatures have been identified that are distinctive of mitochondrial homologs and gram-negative proteobacteria on the one hand and the chloroplasts and cyanobacteria on the other. Detailed phylogenetic analyses of HSP70 sequences from various eubacteria and eukaryotic organellar and cytosolic homologs support the inference regarding the origin of mitochondria from a member of the alpha-proteobacteria and of chloroplasts from cyanobacteria. The analysis presented here also suggests a monophyletic origin of the mitochondrial homologs.     

Discontinuous Occurrence of the hsp70 (dnaK) Gene among Archaea and Sequence Features of HSP70 Suggest a Novel Outlook on Phylogenies Inferred from This Protein

Occurrence of the hsp70 (dnaK) gene was investigated in various members of the domain Archaea comprising both euryarchaeotes and crenarchaeotes and in the hyperthermophilic bacteria Aquifex pyrophilus and Thermotoga maritima representing the deepest offshoots in phylogenetic trees of bacterial 16S rRNA sequences. The gene was not detected in 8 of 10 archaea examined but was found in A. pyrophilus and T. maritima, from which it was cloned and sequenced. Comparative analyses of the HSP70 amino acid sequences encoded in these genes, and others in the databases, showed that (i) in accordance with the vicinities seen in rRNA-based trees, the proteins from A. pyrophilus and T. maritima form a thermophilic cluster with that from the green nonsulfur bacterium Thermomicrobium roseum and are unrelated to their counterparts from gram-positive bacteria, proteobacteria/mitochondria, chlamydiae/spirochetes, deinococci, and cyanobacteria/chloroplasts; (ii) the T. maritima HSP70 clusters with the homologues from the archaea Methanobacterium thermoautotrophicum and Thermoplasma acidophilum, in contrast to the postulated unique kinship between archaea and gram-positive bacteria; and (iii) there are exceptions to the reported association between an insert in HSP70 and gram negativity, or vice versa, absence of insert and gram positivity. Notably, the HSP70 from T. maritima lacks the insert, although T. maritima is phylogenetically unrelated to the gram-positive bacteria. These results, along with the absence of hsp70 (dnaK) in various archaea and its presence in others, suggest that (i) different taxa retained either one or the other of two hsp70 (dnaK) versions (with or without insert), regardless of phylogenetic position; and (ii) archaea are aboriginally devoid of hsp70 (dnaK), and those that have it must have received it from phylogenetically diverse bacteria via lateral gene transfer events that did not involve replacement of an endogenous hsp70 (dnaK) gene.     

Target molecules of molecular chaperone (HSP70 family) in injured gastric mucosa in vivo

AimsSeveral recent studies, including ours, have indicated the importance of heat shock proteins (HSPs) in cytoprotection against cytotoxic agents and environmental stresses mediated by the chaperone function of HSPs (molecular chaperones). However, the target molecule that is recognized by HSPs in damaged cells currently remains unknown. As HSPs rapidly recognize and bind to degenerated protein in cells, target molecules of HSPs might be key molecules for the initiation and pathogenesis of cellular damage. In the present study, gastric mucosal proteins that specifically bind to the HSP70 family (HSC70) were analyzed using HSC70-affinity chromatography.Main methodsThe gastric mucosa was removed from Sprague–Dawley rats after exposure to water immersion-stress for 0, 1, 3 or 5 h. Soluble fractions of each gastric mucosa were applied to the HSC70-affinity column separately. After washing off non-specific binding proteins, specific binding proteins were eluted by ATP-containing buffer. Binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. In addition, the amino acid sequence of purified proteins was also analyzed.Key findingsSpecific HSC70-binding proteins with a molecular weight of 200-kDa and 45-kDa were eluted from an affinity column when gastric mucosal homogenate of 1-h stress exposure was applied. The amino acid sequencing showed that these binding proteins were cytoskeletal myosin (heavy chain) and actin, respectively.SignificanceDuring the pathogenesis of stress-induced gastric mucosal damage, structurally degenerated cytoskeletal myosin (heavy chain) and actin may be key or initiation molecules which structural changes were firstly recognized by molecular chaperone.     

The hsp90 cochaperone p23 is the limiting component of the multiprotein hsp90/hsp70-based chaperone system in vivo where it acts to stabilize the client protein: hsp90 complex

A variety of signaling proteins form heterocomplexes with and are regulated by the heat shock protein chaperone hsp90. These complexes are formed by a multiprotein machinery, including hsp90 and hsp70 as essential and abundant components and Hop, hsp40, and p23 as non-essential cochaperones that are present in much lower abundance in cells. Overexpression of signaling proteins can overwhelm the capacity of this machinery to properly assemble heterocomplexes with hsp90. Here, we show that the limiting component of this assembly machinery in vitro in reticulocyte lysate and in vivo in Sf9 cells is p23. Only a fraction of glucocorticoid receptors (GR) overexpressed in Sf9 cells are in heterocomplex with hsp90 and have steroid binding activity, with the majority of the receptors present as both insoluble and cytosolic GR aggregates. Coexpression of p23 with the GR increases the proportion of cytosolic receptors that are in stable GR.hsp90 heterocomplexes with steroid binding activity, a strictly hsp90-dependent activity for the GR. Coexpression of p23 eliminates the insoluble GR aggregates and shifts the cytosolic receptor from very large aggregates without steroid binding activity to approximately 600-kDa heterocomplexes with steroid binding activity. These data lead us to conclude that p23 acts in vivo to stabilize hsp90 binding to client protein.     

Mouse and Drosophila genes encoding the major heat shock protein (hsp70) are highly conserved.

We used a cloned Drosophila melanogaster hsp70 gene to hybrid-select heat shock-induced mouse mRNA and showed that this mRNA encodes the major mouse heat shock protein. This result suggests that the sequence of the hsp70 gene(s) is highly conserved.