Intermembrane Compartment Research Articles

The Role of the Gravitational Field in Generating Electric Potentials in a Double-Membrane System for Concentration Polarization Conditions.

Electric potentials referred to as the gravielectric effect (∆ΨS) are generated in a double-membrane system containing identical polymer membranes set in horizontal planes and separating non-homogenous electrolyte solutions. The gravielectric effect depends on the concentration and composition of the solutions and is formed due to the gravitational field breaking the symmetry of membrane complexes/concentration boundary layers formed under concentration polarization conditions. As a part of the Kedem-Katchalsky formalism, a model of ion transport was developed, containing the transport parameters of membranes and solutions and taking into account hydrodynamic (convective) instabilities. The transition from non-convective to convective or vice versa can be controlled by a dimensionless concentration polarization factor or concentration Rayleigh number. Using the original measuring set, the time dependence of the membrane potentials was investigated. For steady states, the ∆ΨS was calculated and then the concentration characteristics of this effect were determined for aqueous solutions of NaCl and ethanol. The results obtained from the calculations based on the mathematical model of the gravitational effect are consistent with the experimental results within a 7% error range. It has been shown that a positive or negative gravielectric effect appeared when a density of the solution in the inter-membrane compartment was higher or lower than the density in the outer compartments. The values of the ∆ΨS were in a range from 0 to 27 mV. It was found that, the lower the concentration of solutions in the outer compartments of the two-membrane system (C0), for the same values of Cm/C0, the higher the ∆ΨS, which indicates control properties of the double-membrane system. The considered two-membrane electrochemical system is a source of electromotive force and functions as an electrochemical gravireceptor.

Functional compartmentalization and metabolic separation in a prokaryotic cell

The prokaryotic cell is traditionally seen as a "bag of enzymes," yet its organization is much more complex than in this simplified view. By now, various microcompartments encapsulating metabolic enzymes or pathways are known for Bacteria These microcompartments are usually small, encapsulating and concentrating only a few enzymes, thus protecting the cell from toxic intermediates or preventing unwanted side reactions. The hyperthermophilic, strictly anaerobic Crenarchaeon Ignicoccus hospitalis is an extraordinary organism possessing two membranes, an inner and an energized outer membrane. The outer membrane (termed here outer cytoplasmic membrane) harbors enzymes involved in proton gradient generation and ATP synthesis. These two membranes are separated by an intermembrane compartment, whose function is unknown. Major information processes like DNA replication, RNA synthesis, and protein biosynthesis are located inside the "cytoplasm" or central cytoplasmic compartment. Here, we show by immunogold labeling of ultrathin sections that enzymes involved in autotrophic CO2 assimilation are located in the intermembrane compartment that we name (now) a peripheric cytoplasmic compartment. This separation may protect DNA and RNA from reactive aldehydes arising in the I. hospitalis carbon metabolism. This compartmentalization of metabolic pathways and information processes is unprecedented in the prokaryotic world, representing a unique example of spatiofunctional compartmentalization in the second domain of life.

Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics of Ignicoccus Species.

In this study, the ATP synthase of Ignicoccus hospitalis was purified, characterized, and structurally compared to the respective enzymes of the other Ignicoccus species, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genus Ignicoccus comprises three described species, i.e., I. hospitalis and Ignicoccus islandicus from hot marine sediments near Iceland and Ignicoccus pacificus from a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC). I. hospitalis is the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeon Nanoarchaeum equitansI. hospitalis grows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome of I. hospitalis encodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximal in vitro activity of the I. hospitalis enzyme was measured around pH 6, the optimal stability of the A1AO complex seemed to be at pH 9. Interestingly, the soluble A1 subcomplexes of the different Ignicoccus species exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCE The Crenarchaeota represent one of the major phyla within the Archaea domain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of the Crenarchaeota until now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subject I. hospitalis has a particular importance among crenarchaeotes, since it is the only known host of N. equitans The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.

A Complex Endomembrane System in the Archaeon Ignicoccus hospitalis Tapped by Nanoarchaeum equitans.

Based on serial sectioning, focused ion beam scanning electron microscopy (FIB/SEM), and electron tomography, we depict in detail the highly unusual anatomy of the marine hyperthermophilic crenarchaeon, Ignicoccus hospitalis. Our data support a complex and dynamic endomembrane system consisting of cytoplasmic protrusions, and with secretory function. Moreover, we reveal that the cytoplasm of the putative archaeal ectoparasite Nanoarchaeum equitans can get in direct contact with this endomembrane system, complementing and explaining recent proteomic, transcriptomic and metabolomic data on this inter-archaeal relationship. In addition, we identified a matrix of filamentous structures and/or tethers in the voluminous inter-membrane compartment (IMC) of I. hospitalis, which might be responsible for membrane dynamics. Overall, this unusual cellular compartmentalization, ultrastructure and dynamics in an archaeon that belongs to the recently proposed TACK superphylum prompts speculation that the eukaryotic endomembrane system might originate from Archaea.

The Iho670 fibers of Ignicoccus hospitalis are anchored in the cell by a spherical structure located beneath the inner membrane.

The Iho670 fibers of the hyperthermophilic crenarchaeon of Ignicoccus hospitalis were shown to contain several features that indicate them as type IV pilus-like structures. The application of different visualization methods, including electron tomography and the reconstruction of a three-dimensional model, enabled a detailed description of a hitherto undescribed anchoring structure of the cell appendages. It could be identified as a spherical structure beneath the inner membrane. Furthermore, pools of the fiber protein Iho670 could be localized in the inner as well as the outer cellular membrane of I. hospitalis cells and in the tubes/vesicles in the intermembrane compartment by immunological methods.

Three multihaem cytochromes c from the hyperthermophilic archaeon Ignicoccus hospitalis: purification, properties and localization

Three different multihaem cytochromes c were purified from cell extracts of the hyperthermophilic archaeon Ignicoccus hospitalis. One tetrahaem cytochrome, locus tag designation Igni_0530, was purified from membrane fractions together with the iron-sulfur protein Igni_0529. Two octahaem cytochromes, Igni_0955 and Igni_1359, were purified from soluble fractions but were also present in the membrane fraction. N-terminal sequencing showed that three of the four proteins had their signal peptides cleaved off, while results were ambiguous for Igni_0955. In contrast, mass spectrometry of Igni_0955 and Igni_1359 resulted in single mass peaks including the signal sequences and eight haems per subunit and so both forms might be present in the cell. Igni_0955 and Igni_1359 belong to the hydroxylamine dehydrogenase (HAO) family (29 % mutual identity). HAO or reductase activities with inorganic sulfur compounds were not detected. Igni_0955 was reduced by enriched I. hospitalis hydrogenase at a specific activity of 243 nmol min(-1) (mg hydrogenase)(-1) while activity was non-existent for Igni_0530 and low for Igni_1359. Immuno-electron microscopy of ultra-thin sections showed that Igni_0955 and Igni_1359 are located in both I. hospitalis membranes and also in the intermembrane compartment. We concluded that these cytochromes might function as electron shuttles between the hydrogenase in the outer cellular membrane and cellular reductases, whereas Igni_0530 might be part of the sulfur-reducing mechanism.

The unusual cell biology of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis

The Crenarchaeon Ignicoccus hospitalis is an anaerobic, obligate chemolithoautotrophic hyperthermophile, growing by reduction of elemental sulfur using molecular hydrogen as electron donor. Together with Nanoarchaeum equitans it forms a unique, archaeal biocoenosis, in which I. hospitalis serves as host for N. equitans. Both organisms can be cultivated in a stable coculture which is mandatory for N. equitans but not for I. hospitalis. This strong dependence is affirmed by the fact that N. equitans obtains its lipids and amino acids from the host. I. hospitalis cells exhibit several unique features: they can adhere to surfaces by extracellular appendages ('fibers') which are not used for motility; they use a novel CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate pathway; and they exhibit a unique cell envelope for Archaea consisting of two membranes but lacking an S-layer. These membranes form two cell compartments, a tightly packed cytoplasm surrounded by a weakly staining intermembrane compartment (IMC) with a variable width from 20 to 1,000 nm. In this IMC, many round or elongated vesicles are found which may function as carriers of lipids or proteins out of the cytoplasm. Based on immuno-EM analyses and immuno-fluorescence experiments it was demonstrated recently that the A(1)A(O) ATP synthase, the H(2):sulfur oxidoreductase complex and the acetyl-CoA synthetase (ACS) of I. hospitalis are located in its outermost membrane. Therefore, this membrane is energized and is here renamed as "outer cellular membrane" (OCM). Among all prokaryotes possessing two membranes in their cell envelope, I. hospitalis is the first organism with an energized outermost membrane and ATP synthesis outside the cytoplasm. Since DNA and ribosomes are localized in the cytoplasm, energy conservation is separated from information processing and protein biosynthesis in I. hospitalis. This raises questions concerning the function and characterization of the two membranes, the two cell compartments and of a possible ATP transfer to N. equitans.

AMP-Forming Acetyl Coenzyme A Synthetase in the Outermost Membrane of the Hyperthermophilic Crenarchaeon Ignicoccus hospitalis

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic crenarchaeon was found to possess a new CO(2) fixation pathway, the dicarboxylate/4-hydroxybutyrate cycle. The primary acceptor molecule for this pathway is acetyl coenzyme A (acetyl-CoA), which is regenerated in the cycle via the characteristic intermediate 4-hydroxybutyrate. In the presence of acetate, acetyl-CoA can alternatively be formed in a one-step mechanism via an AMP-forming acetyl-CoA synthetase (ACS). This enzyme was identified after membrane preparation by two-dimensional native PAGE/SDS-PAGE, followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry and N-terminal sequencing. The ACS of I. hospitalis exhibits a molecular mass of ∼690 kDa with a monomeric molecular mass of 77 kDa. Activity tests on isolated membranes and bioinformatic analyses indicated that the ACS is a constitutive membrane-associated (but not an integral) protein complex. Unexpectedly, immunolabeling on cells of I. hospitalis and other described Ignicoccus species revealed that the ACS is localized at the outermost membrane. This perfectly coincides with recent results that the ATP synthase and the H(2):sulfur oxidoreductase complexes are also located in the outermost membrane of I. hospitalis. These results imply that the intermembrane compartment of I. hospitalis is not only the site of ATP synthesis but may also be involved in the primary steps of CO(2) fixation.

Jean‐Marc Reyrat (29/04/1967–28/10/2009)

Jean‐Marc Reyrat (29/04/1967–28/10/2009)

Energized outer membrane and spatial separation of metabolic processes in the hyperthermophilic Archaeon Ignicoccus hospitalis

ATP synthase catalyzes ATP synthesis at the expense of an electrochemical ion gradient across a membrane that can be generated by different exergonic reactions. Sulfur reduction is the main energy-yielding reaction in the hyperthermophilic strictly anaerobic Crenarchaeon Ignicoccus hospitalis. This organism is unusual in having an inner and an outer membrane that are separated by a huge intermembrane compartment. Here we show, on the basis of immuno-EM analyses of ultrathin sections and immunofluorescence experiments with whole I. hospitalis cells, that the ATP synthase and H(2):sulfur oxidoreductase complexes of this organism are located in the outer membrane. These two enzyme complexes are mandatory for the generation of an electrochemical gradient and for ATP synthesis. Thus, among all prokaryotes possessing two membranes in their cell envelope (including Planctomycetes, gram-negative bacteria), I. hospitalis is a unique organism, with an energized outer membrane and ATP synthesis within the periplasmic space. In addition, DAPI staining and EM analyses showed that DNA and ribosomes are localized in the cytoplasm, leading to the conclusion that in I. hospitalis energy conservation is separated from information processing and protein biosynthesis. This raises questions regarding the function of the two membranes, the interaction between these compartments, and the general definition of a cytoplasmic membrane.

Penicillin-binding protein SpoVD disulphide is a target for StoA inBacillus subtilisforespores

The bacterial endospore is a dormant and heat-resistant form of life. StoA (SpoIVH) in Bacillus subtilis is a membrane-bound thioredoxin-like protein involved in endospore cortex synthesis. It is proposed to reduce disulphide bonds in hitherto unknown proteins in the intermembrane compartment of developing forespores. Starting with a bioinformatic analysis combined with mutant studies we identified the sporulation-specific, high-molecular-weight, class B penicillin-binding protein SpoVD as a putative target for StoA. We then demonstrate that SpoVD is a membrane-bound protein with two exposed redox-active cysteine residues. Structural modelling of SpoVD, based on the well characterized orthologue PBP2x of Streptococcus pneumoniae, confirmed that a disulphide bond can form close to the active site of the penicillin-binding domain restricting access of enzyme substrate or functional association with other cortex biogenic proteins. Finally, by exploiting combinations of mutations in the spoVD, stoA and ccdA genes in B. subtilis cells, we present strong in vivo evidence that supports the conclusion that StoA functions to specifically break the disulphide bond in the SpoVD protein in the forespore envelope. The findings contribute to our understanding of endospore biogenesis and open a new angle to regulation of cell wall synthesis and penicillin-binding protein activity.

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein: Is PAM involved in the capacitative calcium entry?

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca 2+ signalling and maintenance of Ca 2+ homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca 2+-ATPase, Na +, K +-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca 2+ ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca 2+ entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca 2+ entry, and their formation and rebuilding have an important regulatory role in cellular Ca 2+ homeostasis.

The mammalian serine protease LACTB is located in the mitochondrial intermembrane compartment

The mammalian serine protease LACTB is located in the mitochondrial intermembrane compartment

Effect of concentration boundary layers on mechanical pressure in a double-membrane system

A mechanical pressure model equations for a double-membrane system, in which a series of two (M l and M r) vertically mounted, flat, symmetric, polymer membranes separate three compartments (l, m, r) containing the heterogeneous non-electrolyte solutions of concentrations C k l, C k m and C k r was developed. These concentrations in initial moment fulfilled the condition C k l < C k m < C k r. The inter-membrane compartment (m) consists of an infinitesimal layer of solution. The volume of the compartments (l, m, r) fulfill the consists on V l= V r 170 V m. The experimental test were performed for binary (aqueous solutions of glucose or ethanol) or ternary (glucose solutions in 0.75 mol·l −1 solution of ethanol or ethanol solutions in 0.1 mol·l −1 aqueous solution of glucose) solutions. The linear dependencies of the mechanical pressure on the concentration difference in binary solutions and non-linear in ternary solutions were obtained. It is shown that the double-membrane system has amplifying properties of mechanical pressure in the intermembrane compartment of a double-membrane system. The experimental results were interpreted in terms of convective instability, which increases the value of solute permeability coefficients of the system: concentration boundary layer/membrane/concentration boundary layer.

Irreversible thermodynamics model equations for heterogeneous solute flows in a double-membrane system

A solute flow model of the equations for a double-membrane system, in which a series of two (Ml and Mr) vertically mounted flat, microporous and symmetric polymer membranes separate three compartments (l, m, r) containing the non-homogeneous, non-ionic solutions was developed. Solution concentrations fulfilled the condition Ckl > Ckm > Ckr. The intermembrane compartment (m) consists of an infinitesimally layer of solution. The volume of the compartments (l, m, r) fulfill the condition Vl= Vr ≈ 170Vm. The experimental tests were performed for binary (aqueous solutions of glucose or ethanol) or ternary (glucose solutions in 0.75 mol·1−1 solution of ethanol or ethanol solutions in 0.1 mol·1−1 aqueous solution of glucose) solutions. The linear dependencies of the solute flux on the concentration difference in binary solutions and non-linear — in ternary solutions — were obtained. It is shown that the double-membrane system has amplifying properties of solute flows. The results obtained during experiments were interpreted in the categories of convective instability, which increased the value of diffusive permeability coefficient of the system: concentration boundary layer/membrane/ concentration boundary layer.

Study of the solute flows of multicomponent and heterogeneous non-ionic solutions in double-membrane system.

The solute flows were studied in a double-membrane osmotic-diffusive cell, in which two membranes mounted in horizontal planes separate three compartments (l,m,r) containing the non-homogeneous, non-electrolytic binary and ternary solutions. The volume of inter-membrane compartment (m), which is the infinitesimally layer of solution, and volume of external compartments (l and r) fulfill the conditions V(m)→ 0 and V(l) =V(r)→ ∞, respectively. In an initial moment, the solution concentrations satisfy the condition (C(o) (s))(l) < (C(o) (s))(m) >(C(o) (s))(r). The double-membrane osmotic-diffusive cell is composed of two complexes: boundary layer/membrane/boundary layer, mounted in horizontal planes. In the cell, solute flux was measured as a function of concentration and gravitational configuration. The linear dependencies of the solute flux on concentration difference in binary solutions and nonlinear - in ternary solutions were obtained. It was shown that the double-membrane osmotic-diffusive cell has rectifying and amplifying properties of solute flows.

Origin of octameric creatine kinases

Mitochondrial creatine kinase (MiCK) occurs primarily as an octameric form localized in the mitochondrial intermembrane compartment in vertebrate tissues and echinoderm spermatozoa (both deuterostome groups). The octameric quaternary structure is thought to play important functional and enzyme targeting roles. We have found that the spermatozoa of the protostome polychaete Chaetopterus variopedatus contain three distinct isoenzymes of creatine kinase (CK) termed CK1, CK2 and CK3. CK3 appears to be present only in the sperm head/midpiece complex where mitochondria are restricted and has a subunit relative molecular mass ( M r) of 43.4 kDa. Gel permeation chromatography using Superdex 200HR showed that CK3 has a native M r of 344.9 kDa indicating that this enzyme exists as an octamer. Electron micrographs of negatively stained CK3 preparations show structures which are virtually identical to those that have been seen for octameric vertebrate MiCK. The above observations show that CK3 from C. variopedatus displays great similarities to MiCKs from vertebrates and echinoderms. Octamerization of CK is not an advanced feature. The evolution of octameric subunit association is ancient and occurred prior to the divergence of protostomes and deuterostomes.

A model equations of the volume transport of multicomponent and heterogeneous non-ionic solutions in double-membrane system.

The volume flows model equation for a double-membrane system, in which two membranes separate three compartments (l,m,r) containing the heterogeneous, non-ionic n-component solutions is elaborated. In this system the solution concentrations fulfill the condition Clk > Cmk > Crk. The inter-membrane compartment (m) consists of the infinitesimal layer of solution. The volume of compartment m and external compartments (l and r) fulfill the conditions Vm→ 0 and Vl =Vr→∞ respectively. The linear dependences of the volume flux on concentration differences in binary solutions and nonlinear - in ternary solutions, were obtained. This model for binary and ternary non-electrolyte solutions is discussed. It is shown, that the double-membrane system has rectifying and amplifying properties for osmotic transport and mechanical pressure.

Structure of mitochondrial creatine kinase.

Creatine kinase (CK, EC 2.7.3.2), an enzyme important for energy metabolism in cells of high and fluctuating energy requirements, catalyses the reversible transfer of a phosphoryl goup from phosphocreatine to ADP. We have solved the structure of the octameric mitochondrial isoform, Mib-CK, which is located in the intermembrane compartment and along the cristae membranes. Mib-CK consumes ATP produced in the mitochondria for the production of phosphocreatine, which is then exported into the cytosol for fast regeneration of ATP by the cytosolic CK isoforms. The octamer has 422 point-group symmetry, and appears as a cube of side length 93 angstrom with a channel 20 angstrom wide extending along the four-fold axis. Positively charged amino acids at the four-fold faces of the octamer possibly interact with negatively charged mitochondrial membranes. Each monomer consists of a small alpha-helical domain and a large domain containing an eight-stranded antiparallel beta-sheet flanked by seven alpha-helices. The conserved residues of the CK family form a compact cluster that covers the active site between the domains.

Mitochondrial creatine kinase mediates contact formation between mitochondrial membranes.

Purified mitochondrial creatine kinase (Mi-CK) (EC 2.7.3.2) from chicken heart was shown to interact simultaneously with purified inner and outer mitochondrial membranes, thereby creating an intermembrane chondrial membranes, thereby creating an intermembrane were purified from rat liver and thus were fully devoid of Mi-CK. Intermembrane contact formation was demonstrated by measuring the binding of inner membrane vesicles to outer membranes spread at the air-water interface. Mi-CK also mediated intermembrane adhesion when membranes formed with total lipid extracts of both membranes were used, pointing to the role of lipids as potential membrane anchors of Mi-CK in the mitochondrial intermembrane space. Other enzymes of the intermembrane space that (like Mi-CK) are also cationic, as well as cytosolic isoenzymes of creatine kinase, failed to induce contact formation. Thus, of the proteins tested, membrane contact formation was specific for Mi-CK. The two oligomeric forms of Mi-CK (octamer and dimer) differed in their ability to mediate intermembrane adhesion, the octamer being more potent. Highly basic peptides, i.e. poly-L-lysines, were shown to strongly interact with membranes formed with lipid extracts of mitochondrial membranes: they both induced intermembrane binding and fusion. Interestingly, the extent of contact formation mediated by poly-L-lysines was lower than that of octameric Mi-CK. The implications of these findings on the function and localization of Mi-CK and on the structure of the mitochondrial intermembrane compartment are discussed.