Tetrameric Protein Complex Research Articles

In Vitro Folding and Assembly of the Escherichia coli ATP-binding Cassette Transporter, BtuCD

Studies on membrane protein folding have focused on monomeric α-helical proteins and a major challenge is to extend this work to larger oligomeric membrane proteins. Here, we study the Escherichia coli (E. coli) ATP-binding cassette (ABC) transporter that imports vitamin B(12) (the BtuCD protein) and use it as a model system for investigating the folding and assembly of a tetrameric membrane protein complex. Our work takes advantage of the modular organization of BtuCD, which consists of two transmembrane protein subunits, BtuC, and two cytoplasmically located nucleotide-binding protein subunits, BtuD. We show that the BtuCD transporter can be re-assembled from both prefolded and partly unfolded, urea denatured BtuC and BtuD subunits. The in vitro re-assembly leads to a BtuCD complex with the correct, native, BtuC and BtuD subunit stoichiometry. The highest rates of ATP hydrolysis were achieved for BtuCD re-assembled from partly unfolded subunits. This supports the idea of cooperative folding and assembly of the constituent protein subunits of the BtuCD transporter. BtuCD folding also provides an opportunity to investigate how a protein that contains both membrane-bound and aqueous subunits coordinates the folding requirements of the hydrophobic and hydrophilic subunits.

Ion Mobility Mass Spectrometry of Two Tetrameric Membrane Protein Complexes Reveals Compact Structures and Differences in Stability and Packing

Here we examined the gas-phase structures of two tetrameric membrane protein complexes by ion mobility mass spectrometry. The collision cross sections measured for the ion channel are in accord with a compact configuration of subunits, suggesting that the native-like structure can be preserved under the harsh activation conditions required to release it from the detergent micelle into the gas phase. We also found that the quaternary structure of the transporter, which has fewer transmembrane subunits than the ion channel, is less stable once stripped of detergents and bulk water. These results highlight the potential of ion mobility mass spectrometry for characterizing the overall topologies of membrane protein complexes and the structural changes associated with nucleotide, lipid, and drug binding.

Implementation of P22 Viral Capsids as Nanoplatforms

Viral capsids are dynamic macromolecular machines which self-assemble and undergo concerted conformational changes during their life cycle. We have taken advantage of the inherent structural flexibility of viral capsids and generated two morphologically different types of viral nanoplatforms from the bacteriophage P22 capsids. Their interior surfaces were genetically manipulated for site-specific attachment of a biotin linker. The extent of internal modifications in each capsid form was characterized by high-resolution mass spectrometry and the analyses revealed that the reactivity of the genetically introduced residues located on the internal surface changes according to the structural transformation of the capsid. Internally modified capsids having 10 nm diameter pores at the 12 icosahedral vertices, so-called wiffle-balls (WB), exhibited the capability to entrap the large tetrameric protein complex streptavidin via the biotin linker anchored onto the interior surface of the WB.

Heteromerization of TRP channel subunits: extending functional diversity

Transient receptor potential (TRP) channels are widely found throughout the animal kingdom. By serving as cellular sensors for a wide spectrum of physical and chemical stimuli, they play crucial physiological roles ranging from sensory transduction to cell cycle modulation. TRP channels are tetrameric protein complexes. While most TRP subunits can form functional homomeric channels, heteromerization of TRP channel subunits of either the same subfamily or different subfamilies has been widely observed. Heteromeric TRP channels exhibit many novel properties compared to their homomeric counterparts, indicating that co-assembly of TRP channel subunits has an important contribution to the diversity of TRP channel functions.

MAP kinase‐activated protein kinases 2 and 3 are required for influenza A virus propagation and act via inhibition of PKR

Influenza viruses have to overcome the type I interferon induced antiviral response to successfully propagate in target cells. A major antiviral factor induced by interferons is the protein kinase R (PKR) that is further activated by dsRNA and phosphorylates the eukaryotic initiation factor 2 (eIF2α). This results in inhibition of protein translation thereby limiting viral replication. Here we describe a novel mechanism by which influenza A viruses escape the antiviral action of PKR. We demonstrate that the mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) MK2 and MK3 are activated on virus infection and, in their active form, directly interact with the repressor of the inhibitor of PKR p88(rIPK). This leads to recruitment of a tetrameric protein complex consisting of p88(rIPK), the inhibitor of PKR p58(IPK) and PKR itself, and finally results in inhibition of the kinase. The importance of MKs for influenza virus propagation was further underscored by demonstrating reduced viral progeny in cells genetically deficient in MK2 or MK3 genes as well as in highly proliferating tumor cells, in which expression of MKs was diminished by specific small interfering RNA. Accordingly, knockdown of MKs resulted in enhanced phosphorylation of PKR and its substrate eIF2α.

The Ets Transcription Factor, GABP, Is Required by Myeloid Progenitors for Normal Myeloid Differentiation.

Abstract 256GABP transcription factor regulates genes that are required for innate immunity. GABP is an obligate tetrameric protein complex that contains two molecules of GABPa, which binds to DNA through its ets domain, and two molecules of GABPb, which contains a transcription activation domain. GABP is an essential component of a multiprotein enhanceosome that is required for retinoic acid-dependent myeloid gene transcription. Disruption in mouse embryo fibroblasts of Gabpa, the mouse gene that encodes mouse Gabpa, causes profound cell cycle arrest at the G1-S boundary, due to reduced expression of DNA Polymerase a and Thymidylate Synthase, which are required for DNA synthesis, and of Skp2, a ubiquitin ligase that controls degradation of the cyclin-dependent kinase inhibitors (CDKIs), p21 and p27. Thus, GABP is a key regulator of the cell cycle. In order to define the role of GABP in myeloid differentiation, we generated mice in which exons that encode the Gabpa ets domain are flanked by loxP recombination sites, and bred these floxed mice to mice that bear the Mx1-Cre transgene. Their progeny were treated with pI-C and Gabpa was efficiently deleted in hematopoietic cells of these Gabpa−/− mice. As controls for all experiments, mice that bear Mx1-Cre but which lack the floxed Gabpa allele were also injected with pI-C. Within days, the peripheral blood white blood cell count fell in the Gabpa−/− mice compared to the controls; half of the Gabpa−/− mice died within two weeks. Gabpa−/− mice exhibited a striking loss of Gr1+, CD11b+ cells in the peripheral blood, spleen, and bone marrow. Myeloid cells of Gabpa−/− mice were immature, morphologically dysplastic, and demonstrated aberrant patterns of myeloid gene expression. Bone marrow from Gabpa−/− mice formed reduced numbers of in vitro myeloid colonies in the presence of G-CSF, M-CSF, or GM-CSF; cells isolated from in vitro colonies from Gabpa−/− mice exhibited a strong bias toward macrophage-like morphology. Multicolor flow cytometry revealed a loss of granulocyte-monocyte committed progenitor cells (GMPs) in the bone marrow of Gabpa−/− mice, and these progenitors expressed aberrant patterns of key transcription factors. Especially notable in Gabpa−/− GMPs was reduced expression of Gfi-1, a transcriptional repressor that is mutated in some congenital neutropenic syndromes, and whose genetic disruption causes abnormalities in granulocyte development. We used chromatin immunoprecipitation (ChIP) to identify ets sites in the Gfi-1 promoter that are bound by GABP in vivo. We conclude that GABP is required for proliferation or survival of committed myeloid progenitor cells and for normal maturation of granulocytes. We hypothesize that defects in myeloid cell proliferation and differentiation associated with Gabpa disruption are caused, at least in part, by its regulation of the Gfi-1 transcriptional repressor. Furthermore, we propose that the regulation of Gfi-1 by GABP constitutes a key regulatory pathway in myeloid cell development. Disclosures:No relevant conflicts of interest to declare.

Calpain Hydrolysis of α- and β2-Adaptins Decreases Clathrin-dependent Endocytosis and May Promote Neurodegeneration

Clathrin-dependent endocytosis is mediated by a tightly regulated network of molecular interactions that provides essential protein-protein and protein-lipid binding activities. Here we report the hydrolysis of the alpha- and beta2-subunits of the tetrameric adaptor protein complex 2 by calpain. Calcium-dependent alpha- and beta2-adaptin hydrolysis was observed in several rat tissues, including brain and primary neuronal cultures. Neuronal alpha- and beta2-adaptin cleavage was inducible by glutamate stimulation and was accompanied by the decreased endocytosis of transferrin. Heterologous expression of truncated forms of the beta2-adaptin subunit significantly decreased the membrane recruitment of clathrin and inhibited clathrin-mediated receptor endocytosis. Moreover, the presence of truncated beta2-adaptin sensitized neurons to glutamate receptor-mediated excitotoxicity. Proteolysis of alpha- and beta2-adaptins, as well as the accessory clathrin adaptors epsin 1, adaptor protein 180, and the clathrin assembly lymphoid myeloid leukemia protein, was detected in brain tissues after experimentally induced ischemia and in cases of human Alzheimer disease. The present study further clarifies the central role of calpain in regulating clathrin-dependent endocytosis and provides evidence for a novel mechanism through which calpain activation may promote neurodegeneration: the sensitization of cells to glutamate-mediated excitotoxicity via the decreased internalization of surface receptors.

Coagonist Release Modulates NMDA Receptor Subtype Contributions at Synaptic Inputs to Retinal Ganglion Cells

NMDA receptors (NMDARs) are tetrameric protein complexes usually comprising two NR1 and two NR2 subunits. Different combinations of four potential NR2 subunits (NR2A-D) confer diversity in developmental expression, subsynaptic localization, and functional characteristics, including affinity for neurotransmitter. NR2B-containing NMDARs, for example, exhibit relatively high affinity both for glutamate and the coagonist glycine. Although multiple NMDAR subtypes can colocalize at individual synapses, particular subtypes often mediate inputs from distinct functional pathways. In retinal ganglion cells (RGCs), NMDARs contribute to synaptic responses elicited by light stimulus onset ("ON") and offset ("OFF"), but roles for particular NMDAR subtypes, and potential segregation between the ON and OFF pathways, have not been explored. Moreover, elements in the retinal circuitry release two different NMDAR coagonists, glycine and d-serine, but the effects of endogenous coagonist release on the relative contribution of different NMDAR subtypes are unclear. Here, we show that coagonist release within the retina modulates the relative contribution of different NMDARs in the ON pathway of the rat retina. By pharmacologically stimulating functional pathways independently in acute slices and recording synaptic responses in RGCs, we show that ON inputs, but not OFF inputs, are mediated in part by NMDARs exhibiting NR2B-like pharmacology. Furthermore, suppressing release of NMDAR coagonist reduces NMDAR activation at ON synapses and increases the relative contribution of these putative NR2B-containing receptors. These results demonstrate direct evidence for evoked coagonist release onto NMDARs and indicate that modulating coagonist release may regulate the relative activation of different NMDAR subtypes in the ON pathway.

The Oligomeric State of the Active BM2 Ion Channel Protein of Influenza B Virus

Influenza A virus and influenza B virus particles both contain small integral membrane proteins (A/M2 and BM2, respectively) that function as a pH-sensitive proton channel and are essential for virus replication. The mechanism of action of the M2 channels is a subject of scientific interest particularly as A/M2 channel was shown to be a target for the action of the antiviral drug amantadine. Unfortunately, an inhibitor of the BM2 channel activity is not known. Thus, knowledge of the structural and functional properties of the BM2 channel is essential for the development of potent antiviral drugs. The characterization of the oligomeric state of the BM2 channel is an essential first step in the understanding of channel function. Here we describe determination of the stoichiometry of the BM2 proton channel by utilizing three different approaches. 1) We demonstrated that BM2 monomers can be chemically cross-linked to yield species consistent with dimers, trimers, and tetramers. 2) We studied electrophysiological and biochemical properties of mixed oligomers consisting of wild-type and mutated BM2 subunits and related these data to predicted binomial distribution models. 3) We used fluorescence resonance energy transfer (FRET) in combination with biochemical measurements to estimate the relationships between BM2 channel subunits expressed in the plasma membrane. Our experimental data are consistent with a tetrameric structure of the BM2 channel. Finally, we demonstrated that BM2 transmembrane domain is responsible for the channel oligomerization.

Low productivity of ribonucleotide reductase in Saccharomyces cerevisiae increases sensitivity to stannous chloride

Ribonucleotide reductase (RNR) of the yeast Saccharomyces cerevisiae is a tetrameric protein complex, consisting of two large and two small subunits. The small subunits Y2 and Y4 form a heterodimer and are encoded by yeast genes RNR2 and RNR4, respectively. Loss of Y4 in yeast mutant rnr4Delta can be compensated for by up-regulated expression of Y2, and the formation of a small subunit Y2Y2 homodimer that allows for a partially functional RNR. However, rnr4Delta mutants exhibit slower growth than wild-type (WT) cells and are sensitive to many mutagens, amongst them UVC and photo-activated mono- and bi-functional psoralens. Cells of the haploid rnr4Delta mutant also show a 3- to 4-fold higher sensitivity to the oxidative stress-inducing chemical stannous chloride than those of the isogenic WT. Both strains acquired increased resistance to SnCl2 with age of culture, i.e., 24-h cultures were more sensitive than cells grown for 2, 3, 4, and 5 days in liquid culture. However, the sensitivity factor of three to four (WT/mutant) did not change significantly. Cultures of the rnr4Delta mutant in stationary phase of growth always showed higher frequency of budding cells (budding index around 0.5) than those of the corresponding WT (budding index <0.1), pointing to a delay of mitosis/cytokinesis.

Formation of NR1/NR2 and NR1/NR3 Heterodimers Constitutes the Initial Step in N-Methyl-D-aspartate Receptor Assembly

N-Methyl-D-aspartate (NMDA) receptors are tetrameric protein complexes composed of the glycine-binding NR1 subunit with a glutamate-binding NR2 and/or glycine-binding NR3 subunit. Tri-heteromeric receptors containing NR1, NR2, and NR3 subunits reconstitute channels, which differ strikingly in many properties from the respective glycine- and glutamate-gated NR1/NR2 complexes and the NR1/NR3 receptors gated by glycine alone. Therefore, an accurate oligomerization process of the different subunits has to assure proper NMDA receptor assembly, which has been assumed to occur via the oligomerization of homodimers. Indeed, using fluorescence resonance energy transfer analysis of differentially fluorescence-tagged subunits and blue native polyacrylamide gel electrophoresis after metabolic labeling and affinity purification revealed that the NR1 subunit is capable of forming homo-oligomeric aggregates. In contrast, both the NR2 and the NR3 subunits formed homo- and hetero-oligomers only in the presence of the NR1 subunit indicating differential roles of the subunits in NMDA receptor assembly. However, co-expression of the NR3A subunit with an N-terminal domain-deleted NR1 subunit (NR1(DeltaNTD)) abrogating NR1 homo-oligomerization did not affect NR1/NR3A receptor stoichiometry or function. Hence, homo-oligomerization of the NR1 subunit is not essential for proper NR1/NR3 receptor assembly. Because identical results were obtained for NR1(DeltaNTD)/NR2 NMDA receptors (Madry, C., Mesic, I., Betz, H., and Laube, B. (2007) Mol. Pharmacol., 72, 1535-1544) and NR1-containing hetero-oligomers are readily formed, we assume that heterodimerization of the NR1 with an NR3 or NR2 subunit, which is followed by the subsequent association of two heterodimers, is the key step in determining proper NMDA receptor subunit assembly and stoichiometry.

Copolymer effects on microglia and T cells in the central nervous system of humanized mice

The random amino acid copolymers FYAK and VWAK ameliorate EAE in a humanized mouse model expressing both a human transgenic myelin basic protein (MBP)85-99-specific T cell receptor and HLA-DR2. Here we show that microglia isolated from the central nervous system (CNS) of humanized mice with EAE induced by MBP85-99 and treated with these copolymers had reduced expression of HLA-DR, and thus reduced capacity to present MBP85-99 and activate transgenic T cells. In vitro microglia up-regulated empty HLA-DR2 upon activation with GM-CSF with or without LPS or IFN-gamma, but not with IL-4 or IL-10. Correspondingly, gene chip arrays showed that the CNS of untreated and YFAK-treated mice differentially expressed pro- and anti-inflammatory molecules during MBP85-99-induced EAE. Interestingly, microglia expressed the full-length gammabeta and alphabeta subunits of the tetrameric adaptor protein complexes AP-1 and AP-2 respectively, but after treatment with GM-CSF these complexes were cleaved, as had been found in immature dendritic cells derived from bone marrow. Strikingly, in vivo the perivascular lymphocyte infiltration seen in untreated mice immunized with MBP85-99 was composed of equal numbers of hVbeta2+ MPB85-99-specific transgenic and hVbeta2- endogenous T cells, while the much smaller infiltration seen after treatment with YFAK was composed predominantly of hVbeta2- endogenous T cells.

Structure and functional characterization of single-strand DNA binding protein SSDP1: Carboxyl-terminal of SSDP1 has transcription activity

LIM-homeodomain transcription factors control a wide range of developmental processes, such as early patterning of the embryonic development, organ formations of brain, limbs, and eyes. Molecular mechanisms of the underlying processes involve complicated multiple protein complexes that direct transcription activation of target genes, protein-protein interactions, and transcriptional regulations. Among those molecules, cofactor Ldb1, interacting with LIM/homeobox family transcription factor, defines a tetrameric protein complex in controlling downstream genes of transcriptional regulation. In addition, SSDP specifically involves this complex supported by showing that both SSDP1 and SSDP2 bind to Ldb1 in vivo. Here it has been found that SSDP1 itself is a transcription factor that has transcription activity independently. Furthermore, C-terminal of SSDP1 possessing an entire transcription activity in vivo, confirmed in both yeast and mammalian cells, has been defined. Interestingly, the transcriptional function of SSDP1 was not required for the interaction with Ldb1. Thus, biochemical data of SSDP1 presented by this study provides biochemical evidence for a better understanding of transcriptional regulation.

Reduced Aph‐1b expression causes tissue‐ and substrate‐specific changes in γ‐secretase activity in rats with a complex phenotype

The gamma-secretase enzyme complex displays intramembrane catalytic activity toward many type I transmembrane proteins, including the Alzheimer-linked amyloid-beta-protein precursor (APP) and the neuregulin receptor ErbB4. Active gamma-secretase is a tetrameric protein complex consisting of presenilin-1 (or -2), nicastrin, PEN-2, and Aph-1a (or -1b). We have recently discovered that pharmacogenetically bred apomorphine-susceptible Wistar rats (APO-SUS) have only one or two copies of the Aph-1b gene (termed I/I and II/II rats, respectively), whereas their phenotypic counterparts (APO-UNSUS) have three copies (III/III). As a result, APO-SUS rats display reduced Aph-1b expression and a complex phenotype reminiscent of neurodevelopmental disorders. Here we determined in the I/I and III/III rats the gamma-secretase cleavage activity toward the three APP superfamily members, p75 neurotrophin receptor, ErbB4, and neuregulin-2, and found that the cleavage of only a subset of the substrates was changed. Furthermore, the observed differences were restricted to tissues that normally express relatively high Aph-1b compared with Aph-1a levels. Thus, we provide in vivo evidence that subtle alterations in gamma-secretase subunit composition may lead to a variety of affected (neuro)developmental signaling pathways and, consequently, a complex phenotype.

Gas-phase dissociation pathways of a tetrameric protein complex

The gas-phase dissociation of the tetrameric complex transthyretin (TTR) has been investigated with tandem-mass spectrometry (tandem-MS) using a nanoflow-electrospray interface and a quadrupole time-of-flight (Q-TOF) mass spectrometer. The results show that highly charged monomeric product ions dissociate from the macromolecular complex to form trimeric products. Manipulating the pressure conditions within the mass spectrometer facilitates the formation of metastable ions. These were observed for the transitions from tetrameric to monomeric and trimeric product ions and additionally for losses of small molecules associated with the protein complex in the gas phase. These results are interpreted in the light of recent mechanisms for the electrospray process and provide insight into the composition and factors governing the stability of macromolecular ions in the gas phase.

Recombinant water-soluble chlorophyll protein from Brassica oleracea var. Botrys binds various chlorophyll derivatives.

A gene coding for water-soluble chlorophyll-binding protein (WSCP) from Brassica oleracea var. Botrys has been used to express the protein, extended by a hexahistidyl tag, in Escherichia coli. The protein has been refolded in vitro to study its pigment binding behavior. Recombinant WSCP was found to bind two chlorophylls (Chls) per tetrameric protein complex but no carotenoids in accordance with previous observations with the native protein [Satoh, H., Nakayama, K., Okada, M. (1998) J. Biol. Chem. 273, 30568-30575]. WSCP binds Chl a, Chl b, bacteriochlorophyll a, and the Zn derivative of Chl a but not pheophytin a, indicating that the central metal ion in Chl is essential for binding. WSCP also binds chlorophyllides a and b and even the more distant Chl precursor Mg-protoporphyrin IX; however, these pigments fail to induce oligomerization of the protein. We conclude that the phytol group in bound Chl plays a role in the formation of tetrameric WSCP complexes. If WSCP in fact binds Chl or its derivative(s) in vivo, the lack of carotenoids in pigmented WSCP raises the question of how photooxidation, mediated by triplet-excited Chl and singlet oxygen, is prohibited. We show by spin-trap electron-paramagnetic resonance that the light-induced singlet-oxygen formation of WSCP-bound Chl is lower by a factor of about 4 than that of unbound Chl. This as-yet-unknown mechanism of WSCP to protect its bound Chl against photooxidation supports the notion that WSCP may function as a transient carrier of Chl or its derivatives.

Functional Expression of a Fusion-dimeric MoFe Protein of Nitrogenase in Azotobacter vinelandii

The MoFe protein component of the complex metalloenzyme nitrogenase is an alpha2beta2 tetramer encoded by the nifD and the nifK genes. In nitrogen fixing organisms, the alpha and beta subunits are translated as separate polypeptides and then assembled into tetrameric MoFe protein complex that includes two types of metal centers, the P cluster and the FeMo cofactor. In Azotobacter vinelandii, the NifEN complex, the site for biosynthesis of the FeMo cofactor, is an alpha2beta2 tetramer that is structurally similar to the MoFe protein and encoded as two separate polypeptides by the nifE and the nifN genes. In Anabaena variabilis it was shown that a NifE-N fusion protein encoded by translationally fused nifE and nifN genes can support biological nitrogen fixation. The structural similarity between the MoFe protein and the NifEN complex prompted us to test whether the MoFe protein could also be functional when synthesized as a single protein encoded by nifD-K translational fusion. Here we report that the NifD-K fusion protein encoded by nifD-K translational fusion in A. vinelandii is a large protein (as determined by Western blot analysis) and is capable of supporting biological nitrogen fixation. These results imply that the MoFe protein is flexible in that it can accommodate major structural changes and remain functional.

Identification of novel protein-protein interactions at the cytosolic surface of the Sec63 complex in the yeast ER membrane.

Precursors of secretory proteins are targeted to the membrane of the endoplasmic reticulum by specific protein complexes that recognize their signal sequence. All eukaryotic cells investigated so far have been found to possess the signal recognition particle (SRP) that targets the majority of precursors to the translocation machinery. In Saccharomyces cerevisiae a number of proteins are translocated independently of SRP. These precursors rely on a different signal sequence-binding complex, which includes Sec62p, Sec63p, Sec71p and Sec72p. Identifying interactions between individual components of this tetrameric protein complex is important in the understanding of its function. We demonstrate a specific interaction between the only two essential proteins in this complex, Sec62p and Sec63p. Second, we show evidence of homodimerization of Sec72p molecules and further identify the YLR301w gene product as a novel in vivo interacting partner of Sec72p. Finally, we determine the authentic N-terminus of Sec62p and describe interacting subdomains of both Sec62p and Sec63p.

Pyrolobus fumarii, gen. and sp. nov., represents a novel group of archaea, extending the upper temperature limit for life to 113 degrees C.

A novel, irregular, coccoid-shaped archaeum was isolated from a hydrothermally heated black smoker wall at the TAG site at the Mid Atlantic Ridge (depth 3650 meters). It grew at between 90 degrees C and 113 degrees C (optimum 106 degrees C) and pH 4.0-6.5 (optimum 5.5) and 1%-4% salt (optimum 1.7%). The organism was a facultatively aerobic obligate chemolithoautotroph gaining energy by H2-oxidation. Nitrate, S2O3(2-), and low concentrations of O2 (up to 0.3% v/v) served as electron acceptors, yielding NH4+, H2S, and H2O as end products, respectively. Growth was inhibited by acetate, pyruvate, glucose, starch, or sulfur. The new isolate was able to form colonies on plates (at 102 degrees C) and to grow at a pressure of 25000 kPa (250 bar). Exponentially growing cultures survived a one-hour autoclaving at 121 degrees C. The GC content was 53 mol%. The core lipids consisted of glycerol-dialkyl glycerol tetraethers and traces of 2,3-di-O-phytanyl-sn-glycerol. The cell wall was composed of a surface layer of tetrameric protein complexes arranged on a p4-lattice (center-to-center distance 18.5 nm). By its 16S rRNA sequence, the new isolate belonged to the Pyrodictiaceae. Based on its GC-content, DNA homology, S-layer composition, and metabolism, we describe here a new genus, which we name Pyrolobus (the "fire lobe"). The type species is Pyrolobus fumarii (type strain 1A; DSM 11204).