Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria.

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane-adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel-fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis, we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large-scale lipid phase separation and protein segregation in intact, protein-crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible.

Alternative Oxidase Isoforms Are Differentially Activated by Tricarboxylic Acid Cycle Intermediates.

The cyanide-insensitive alternative oxidase (AOX) is a non-proton-pumping ubiquinol oxidase that catalyzes the reduction of oxygen to water and is posttranslationally regulated by redox mechanisms and 2-oxo acids. Arabidopsis (Arabidopsis thaliana) possesses five AOX isoforms (AOX1A-AOX1D and AOX2). AOX1D expression is increased in aox1a knockout mutants from Arabidopsis (especially after restriction of the cytochrome c pathway) but cannot compensate for the lack of AOX1A, suggesting a difference in the regulation of these isoforms. Therefore, we analyzed the different AOX isoenzymes with the aim to identify differences in their posttranslational regulation. Seven tricarboxylic acid cycle intermediates (citrate, isocitrate, 2-oxoglutarate, succinate, fumarate, malate, and oxaloacetate) were tested for their influence on AOX1A, AOX1C, and AOX1D wild-type protein activity using a refined in vitro system. AOX1C is insensitive to all seven organic acids, AOX1A and AOX1D are both activated by 2-oxoglutarate, but only AOX1A is additionally activated by oxaloacetate. Furthermore, AOX isoforms cannot be transformed to mimic one another by substituting the variable cysteine residues at position III in the protein. In summary, we show that AOX isoforms from Arabidopsis are differentially fine-regulated by tricarboxylic acid cycle metabolites (most likely depending on the amino-terminal region around the highly conserved cysteine residues known to be involved in regulation by the 2-oxo acids pyruvate and glyoxylate) and propose that this is the main reason why they cannot functionally compensate for each other.

Analysis of Posttranslational Activation of Alternative Oxidase Isoforms.

Mitochondrial alternative oxidase (AOX) in plants is a non-proton-motive ubiquinol oxidase that is activated by redox mechanisms and 2-oxo acids. A comparative analysis of the AOX isoenzymes AOX1A, AOX1C, and AOX1D from Arabidopsis (Arabidopsis thaliana) revealed that cysteine residues, CysI and CysII, are both involved in 2-oxo acid activation, with AOX1A activity being more increased by 2-oxo acids than that of AOX1C and AOX1D. Substitution of cysteine in AOX1A by glutamate mimicked its activation by pyruvate or glyoxylate, but not in AOX1C and AOX1D. CysIII, only present in AOX1A, is not involved in activation by reduction or metabolites, but substitutions at this position affected activity. AOX1A carrying a serine residue at position CysI was activated by succinate, while correspondingly substituted variants of AOX1C and AOX1D were insensitive. Activation by glutamate at CysI and CysII is consistent with the formation of the thiohemiacetal, while succinate activation after changing CysI to serine suggests hemiacetal formation. Surprisingly, in AOX1A, replacement of CysI by alanine, which cannot form a (thio)hemiacetal, led to even higher activities, pointing to an alternative mechanism of activation. Taken together, our results demonstrate that AOX isoforms are differentially activated and that activation at CysI and CysII is additive.

Membrane protein insertion and assembly by the bacterial holo-translocon SecYEG-SecDF-YajC-YidC.

Protein secretion and membrane insertion occur through the ubiquitous Sec machinery. In this system, insertion involves the targeting of translating ribosomes via the signal recognition particle and its cognate receptor to the SecY (bacteria and archaea)/Sec61 (eukaryotes) translocon. A common mechanism then guides nascent transmembrane helices (TMHs) through the Sec complex, mediated by associated membrane insertion factors. In bacteria, the membrane protein 'insertase' YidC ushers TMHs through a lateral gate of SecY to the bilayer. YidC is also thought to incorporate proteins into the membrane independently of SecYEG. Here, we show the bacterial holo-translocon (HTL) - a supercomplex of SecYEG-SecDF-YajC-YidC - is a bona fide resident of the Escherichia coli inner membrane. Moreover, when compared with SecYEG and YidC alone, the HTL is more effective at the insertion and assembly of a wide range of membrane protein substrates, including those hitherto thought to require only YidC.

Refined method to study the posttranslational regulation of alternative oxidases from Arabidopsis thaliana in vitro.

In isolated membranes, posttranslational regulation of quinol oxidase activities can only be determined simultaneously for all oxidases - quinol oxidases as well as cytochrome c oxidases - because of their identical localization. In this study, a refined method to determine the specific activity of a single quinol oxidase is exemplarily described for the alternative oxidase (AOX) isoform AOX1A from Arabidopsis thaliana and its corresponding mutants, using the respiratory chain of an Escherichia coli cytochrome bo and bd-I oxidase double mutant as a source to provide electrons necessary for O2 reduction via quinol oxidases. A highly sensitive and reproducible experimental set-up with prolonged linear time intervals of up to 60 s is presented, which enables the determination of constant activity rates in E. coli membrane vesicles enriched in the quinol oxidase of interest by heterologous expression, using a Clark-type oxygen electrode to continuously follow O2 consumption. For the calculation of specific quinol oxidase activity, activity rates were correlated with quantitative signal intensity determinations of AOX1A present in a membrane-bound state by immunoblot analyses, simultaneously enabling normalization of specific activities between different AOX proteins. In summary, the method presented is a powerful tool to study specific activities of individual quinol oxidases, like the different AOX isoforms, and their corresponding mutants upon modification by addition of effectors/inhibitors, and thus to characterize their individual mode of posttranslational regulation in a membranous environment.

Assembly of the Escherichia coli FoF1 ATP synthase involves distinct subcomplex formation.

The ATP synthase (FoF1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane by Fo to ATP synthesis or hydrolysis in F1. Whereas good knowledge of the nanostructure and the rotary mechanism of the ATP synthase is at hand, the assembly pathway of the 22 polypeptide chains present in a stoichiometry of ab2c10α3ß3γδϵ has so far not received sufficient attention. In our studies, mutants that synthesize different sets of FoF1 subunits allowed the characterization of individually formed stable subcomplexes. Furthermore, the development of a time-delayed in vivo assembly system enabled the subsequent synthesis of particular missing subunits to allow the formation of functional ATP synthase complexes. These observations form the basis for a model that describes the assembly pathway of the E. coli ATP synthase from pre-formed subcomplexes, thereby avoiding membrane proton permeability by a concomitant assembly of the open H+-translocating unit within a coupled FoF1 complex.

Subunit δ is the key player for assembly of the H(+)-translocating unit of Escherichia coli F(O)F1 ATP synthase.

The ATP synthase (F(O)F1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c10γε and the stator ab2α3ß3δ. To study the assembly of this multimeric enzyme complex consisting of membrane-integral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled F(O)F1 complexes by affinity chromatography with the use of mutants synthesizing different sets of F(O)F1 subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that F(O)F1 is assembled in a modular way via subcomplexes, thereby preventing the formation of a functional H(+)-translocating unit as intermediate product. Surprisingly, during the biogenesis of F(O)F1, F1 subunit δ is the key player in generating stable F(O). Subunit δ serves as clamp between ab2 and c10α3ß3γε and guarantees that the open H(+) channel is concomitantly assembled within coupled F(O)F1 to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H(+)-translocating enzymes.

Individual interactions of the b subunits within the stator of the Escherichia coli ATP synthase.

FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit δ in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit δ, whereas the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-α, b-ß, b-δ, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. bI and bII). The results show the b dimer to be located at a non-catalytic α/ß cleft, with bI close to subunit α, whereas bII is proximal to subunit ß. Furthermore, bI can be linked to subunit δ as well as to subunit a. Among the subcomplexes formed were a-bI-α, bII-ß, α-bI-bII-ß, and a-bI-δ. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that bI is functionally related to the single b subunit present in mitochondrial ATP synthase.

Time-delayed in vivo assembly of subunit a into preformed Escherichia coli FoF1 ATP synthase.

Escherichia coli F(O)F(1) ATP synthase, a rotary nanomachine, is composed of eight different subunits in a α3ß3γδεab2c10 stoichiometry. Whereas F(O)F(1) has been studied in detail with regard to its structure and function, much less is known about how this multisubunit enzyme complex is assembled. Single-subunit atp deletion mutants are known to be arrested in assembly, thus leading to formation of partially assembled subcomplexes. To determine whether those subcomplexes are preserved in a stable standby mode, a time-delayed in vivo assembly system was developed. To establish this approach, we targeted the time-delayed assembly of membrane-integrated subunit a into preformed F(O)F(1) lacking subunit a (F(O)F(1)-a) which is known to form stable subcomplexes in vitro. Two expression systems (araBADp and T7p-laco) were adjusted to provide compatible, mutually independent, and sufficiently stringent induction and repression regimens. In detail, all structural atp genes except atpB (encoding subunit a) were expressed under the control of araBADp and induced by arabinose. Following synthesis of F(O)F(1)-a during growth, expression was repressed by glucose/d-fucose, and degradation of atp mRNA controlled by real-time reverse transcription-PCR. A time-delayed expression of atpB under T7p-laco control was subsequently induced in trans by addition of isopropyl-ß-d-thiogalactopyranoside. Formation of fully assembled, and functional, F(O)F(1) complexes was verified. This demonstrates that all subunits of F(O)F(1)-a remain in a stable preformed state capable to integrate subunit a as the last subunit. The results reveal that the approach presented here can be applied as a general method to study the assembly of heteromultimeric protein complexes in vivo.

Dynamics of bioenergetic microcompartments.

The vast majority of life on earth is dependent on harvesting electrochemical potentials over membranes for the synthesis of ATP. Generation of membrane potential often relies on electron transport through membrane protein complexes, which vary among the bioenergetic membranes found in living organisms. In order to maximize the efficient harvesting of the electrochemical potential, energy loss must be minimized, and this is achieved partly by restricting certain events to specific microcompartments, on bioenergetic membranes. In this review we will describe the characteristics of the energy-converting supramolecular structures involved in oxidative phosphorylation in mitochondria and bacteria, and photophosphorylation. Efficient function of electron transfer pathways requires regulation of electron flow, and we will also discuss how this is partly achieved through dynamic re-compartmentation of the membrane complexes into different supercomplexes. In addition to supercomplexes, the supramolecular structure of the membrane, and in particular the role of water layers on the surface of the membrane in the prevention of wasteful proton escape (and therefore energy loss), is discussed in detail. In summary, the restriction of energetic processes to specific microcompartments on bioenergetic membranes minimizes energy loss, and dynamic rearrangement of these structures allows for regulation.

Functional production of the Na+ F1F(O) ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI.

The Na(+) F(1)F(O) ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F(O)V(O) hybrid rotor that contains nine copies of a F(O)-like c subunit and one copy of a V(O)-like c(1) subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na(+) F(1)F(O) ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na(+) F(1)F(O) ATP synthase that was purified in a single step after inserting a His(6)-tag to its ß subunit. The purified enzyme was competent in Na(+) transport and contained the F(O)V(O) hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na(+) F(1)F(O) ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F(O)V(O) hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.

Constant c10 ring stoichiometry in the Escherichia coli ATP synthase analyzed by cross-linking.

The subunit c stoichiometry of Escherichia coli ATP synthase was studied by intermolecular cross-linking via oxidation of bi-cysteine-substituted subunit c (cA21C/cM65C). Independent of the carbon source used for growth and independent of the presence of other FoF1 subunits, an equal pattern of cross-link formation stopping at the formation of decamers was obtained.

The stoichiometry of subunit c of Escherichia coli ATP synthase is independent of its rate of synthesis.

Immunoblot quantitation of Escherichia coli ATP synthase isolated from atp wildtype and mutant cells, the latter comprising a reduced expression of the atpE gene coding for subunit c due to a point mutation within its Shine-Dalgarno sequence, suggested a variable stoichiometry of subunit c [Schemidt et al. (1995) Arch. Biochem. Biophys. 323, 423-428]. To study the c ring of the mutant strain and its stoichiometry in more detail, F O isolated from wildtype and mutant were investigated by quantitation, reconstitution, and cross-linking. Direct quantitation by staining with SYPRO Ruby revealed a reduction of subunit c in the mutant by a factor of 2 compared to F O subunits a and b. Rates of passive H (+) translocation correlated with the amount of subunit c present. Lower rates for mutant F O could be increased by addition of subunit c, whereas translocation rates remained constant by coreconstitution with nonfunctional subunit cD61G arguing against the presence of smaller c rings that are filled up with coreconstituted subunit c. Intermolecular cross-linking by oxidation of bicysteine-substituted subunit c ( cA21C/ cM65C) revealed an equal pattern of oligomer formation in wildtype and mutant also favoring a comparable subunit c stoichiometry. Cross-linking of membrane vesicles containing cysteine-substituted subunits a ( aN214C) and c ( cM65C) characterized the mutant F O preparation as a heterogeneous population, which consists of assembled F O and free ab 2 subcomplexes each present to approximately 50%. Thus, these data clearly demonstrate that the stoichiometry of the subunit c rings remains constant even after reduction of the synthesis of subunit c.

The holo-form of the nucleotide binding domain of the KdpFABC complex from Escherichia coli reveals a new binding mode.

P-type ATPases are ubiquitously abundant enzymes involved in active transport of charged residues across biological membranes. The KdpB subunit of the prokaryotic Kdp-ATPase (KdpFABC complex) shares characteristic regions of homology with class II-IV P-type ATPases and has been shown previously to be misgrouped as a class IA P-type ATPase. Here, we present the NMR structure of the AMP-PNP-bound nucleotide binding domain KdpBN of the Escherichia coli Kdp-ATPase at high resolution. The aromatic moiety of the nucleotide is clipped into the binding pocket by Phe(377) and Lys(395) via a pi-pi stacking and a cation-pi interaction, respectively. Charged residues at the outer rim of the binding pocket (Arg(317), Arg(382), Asp(399), and Glu(348)) stabilize and direct the triphosphate group via electrostatic attraction and repulsion toward the phosphorylation domain. The nucleotide binding mode was corroborated by the replacement of critical residues. The conservative mutation F377Y produced a high residual nucleotide binding capacity, whereas replacement by alanine resulted in low nucleotide binding capacities and a considerable loss of ATPase activity. Similarly, mutation K395A resulted in loss of ATPase activity and nucleotide binding affinity, even though the protein was properly folded. We present a schematic model of the nucleotide binding mode that allows for both high selectivity and a low nucleotide binding constant, necessary for the fast and effective turnover rate realized in the reaction cycle of the Kdp-ATPase.

Direct interaction of subunits a and b of the F0 complex of Escherichia coli ATP synthase by forming an ab2 subcomplex.

The addition of a His6 tag to the N terminus of subunit a of the F0 complex of the Escherichia coli ATP synthase allowed the purification of an ab2 subcomplex after solubilization of membranes with n-dodecyl-beta-d-maltoside and subsequent nickel-nitrilotriacetic acid affinity chromatography. After co-reconstitution of the ab2 subcomplex with purified subunit c, passive proton translocation rates as well as coupled ATPase activities after binding of F1 were measured that were comparable with those of wild type F0. The interaction between subunits a and b, which has been shown to be stoichiometric and functional, is not triggered by any cross-linking reagent and therefore reflects subunit interactions occurring within the F0 complex in vivo.