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1.  Intracytoplasmic Copper Homeostasis Controls Cytochrome c Oxidase Production 
mBio  2014;5(1):e01055-13.
ABSTRACT
Copper is an essential micronutrient used as a metal cofactor by a variety of enzymes, including cytochrome c oxidase (Cox). In all organisms from bacteria to humans, cellular availability and insertion of copper into target proteins are tightly controlled due to its toxicity. The major subunit of Cox contains a copper atom that is required for its catalytic activity. Previously, we identified CcoA (a member of major facilitator superfamily transporters) as a component required for cbb3-type Cox production in the Gram-negative, facultative phototroph Rhodobacter capsulatus. Here, first we demonstrate that CcoA is a cytoplasmic copper importer. Second, we show that bypass suppressors of a ccoA deletion mutant suppress cbb3-Cox deficiency by increasing cellular copper content and sensitivity. Third, we establish that these suppressors are single-base-pair insertion/deletions located in copA, encoding the major P1B-type ATP-dependent copper exporter (CopA) responsible for copper detoxification. A copA deletion alone has no effect on cbb3-Cox biogenesis in an otherwise wild-type background, even though it rescues the cbb3-Cox defect in the absence of CcoA and renders cells sensitive to copper. We conclude that a hitherto unknown functional interplay between the copper importer CcoA and the copper exporter CopA controls intracellular copper homeostasis required for cbb3-Cox production in bacteria like R. capsulatus.
IMPORTANCE
Copper (Cu) is an essential micronutrient required for many processes in the cell. It is found as a cofactor for heme-copper containing cytochrome c oxidase enzymes at the terminus of the respiratory chains of aerobic organisms by catalyzing reduction of dioxygen (O2) to water. Defects in the biogenesis and copper insertion into cytochrome c oxidases lead to mitochondrial diseases in humans. This work shows that a previously identified Cu transporter (CcoA) is a Cu importer and illustrates the link between two Cu transporters, the importer CcoA and the exporter CopA, required for Cu homeostasis and Cu trafficking to cytochrome c oxidase in the cell.
doi:10.1128/mBio.01055-13
PMCID: PMC3903287  PMID: 24425735
2.  The ScoI homologue SenC is a copper binding protein that interacts directly with the cbb3-type cytochrome oxidase in Rhodobacter capsulatus 
Biochimica et biophysica acta  2012;1817(11):2005-2015.
Sco proteins are widespread assembly factors for the CuA centre of aa3-type cytochrome oxidases in eukaryotic and prokaryotic organisms. However, Sco homologues are also found in bacteria like Rhodobacter capsulatus which lack aa3-type cytochrome oxidases and instead use a cbb3-type cytochrome oxidase (cbb3 Cox) without a CuA centre as a terminal oxidase. In the current study, we have analyzed the role of Sco (SenC) during cbb3 Cox assembly in Rhodobacter capsulatus. In agreement with earlier works, we found a strong cbb3 Cox defect in the absence of SenC that impairs the steady-state stability of the CcoN, CcoO and CcoP core subunits, without the accumulation of detectable assembly intermediates. In vivo cross-linking results demonstrate that SenC is in close proximity to the CcoP and CcoH subunits of cbb3 Cox, suggesting that SenC interacts directly with cbb3 Cox during its assembly. SenC binds copper and the cbb3 Cox assembly defect in the absence of SenC can be rescued by the addition of least 0.5 μM Cu. Neither copper nor SenC influenced the transcription of the ccoNOQP operon encoding for cbb3 Cox. Transcription of senC itself was also not influenced by Cu unless the putative Cu-export ATPase CcoI was absent. As CcoI is specifically required for the cbb3 Cox assembly, these data provide a direct link between Cu delivery to cbb3 Cox and SenC function.
doi:10.1016/j.bbabio.2012.06.621
PMCID: PMC3432722  PMID: 22771512
Sco proteins; copper insertion; cytochrome c oxidase; copper transport; respiratory growth; Rhodobacter capsulatus
3.  Missense Mutations in Cytochrome c Maturation Genes Provide New Insights into Rhodobacter capsulatus cbb3-Type Cytochrome c Oxidase Biogenesis 
Journal of Bacteriology  2013;195(2):261-269.
The Rhodobacter capsulatus cbb3-type cytochrome c oxidase (cbb3-Cox) belongs to the heme-copper oxidase superfamily, and its subunits are encoded by the ccoNOQP operon. Biosynthesis of this enzyme is complex and needs dedicated biogenesis genes (ccoGHIS). It also relies on the c-type cytochrome maturation (Ccm) process, which requires the ccmABCDEFGHI genes, because two of the cbb3-Cox subunits (CcoO and CcoP) are c-type cytochromes. Recently, we reported that mutants lacking CcoA, a major facilitator superfamily type transporter, produce very small amounts of cbb3-Cox unless the growth medium is supplemented with copper. In this work, we isolated “Cu-unresponsive” derivatives of a ccoA deletion strain that exhibited no cbb3-Cox activity even upon Cu supplementation. Molecular characterization of these mutants revealed missense mutations in the ccmA or ccmF gene, required for the Ccm process. As expected, Cu-unresponsive mutants lacked the CcoO and CcoP subunits due to Ccm defects, but remarkably, they contained the CcoN subunit of cbb3-Cox. Subsequent construction and examination of single ccm knockout mutants demonstrated that membrane insertion and stability of CcoN occurred in the absence of the Ccm process. Moreover, while the ccm knockout mutants were completely incompetent for photosynthesis, the Cu-unresponsive mutants grew photosynthetically at lower rates and produced smaller amounts of cytochromes c1 and c2 than did a wild-type strain due to their restricted Ccm capabilities. These findings demonstrate that different levels of Ccm efficiency are required for the production of various c-type cytochromes and reveal for the first time that maturation of the heme-Cu-containing subunit CcoN of R. capsulatus cbb3-Cox proceeds independently of that of the c-type cytochromes during the biogenesis of this enzyme.
doi:10.1128/JB.01415-12
PMCID: PMC3553827  PMID: 23123911
4.  Biogenesis of cbb3-type cytochrome c oxidase in Rhodobacter capsulatus 
Biochimica et Biophysica Acta  2011;1817(6):898-910.
The cbb3-type cytochrome c oxidases (cbb3-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa3-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixation. In addition, they are prevalent in many pathogenic bacteria, and important for colonizing low oxygen tissues. Studies related to cbb3-Cox provide a fascinating paradigm for the biogenesis of sophisticated oligomeric membrane proteins. Complex subunit maturation and assembly machineries, producing the c-type cytochromes and the binuclear heme b3-CuB center, have to be coordinated precisely both temporally and spatially to yield a functional cbb3-Cox enzyme. In this review we summarize our current knowledge on the structure, regulation and assembly of cbb3-Cox, and provide a highly tentative model for cbb3-Cox assembly and formation of its heme b3-CuB binuclear center.
doi:10.1016/j.bbabio.2011.10.011
PMCID: PMC3304015  PMID: 22079199
cbb3-type cytochrome c oxidase; co- or post-translational cofactor insertion; assembly of membrane proteins; copper acquisition; photosynthesis and respiration; energy transduction
5.  Propionic acidemia: clinical course and outcome in 55 pediatric and adolescent patients 
Background
Propionic acidemia is an inherited disorder caused by deficiency of propionyl-CoA carboxylase. Although it is one of the most frequent organic acidurias, information on the outcome of affected individuals is still limited.
Study design/methods
Clinical and outcome data of 55 patients with propionic acidemia from 16 European metabolic centers were evaluated retrospectively. 35 patients were diagnosed by selective metabolic screening while 20 patients were identified by newborn screening. Endocrine parameters and bone age were evaluated. In addition, IQ testing was performed and the patients’ and their families’ quality of life was assessed.
Results
The vast majority of patients (>85%) presented with metabolic decompensation in the neonatal period. Asymptomatic individuals were the exception. About three quarters of the study population was mentally retarded, median IQ was 55. Apart from neurologic symptoms, complications comprised hematologic abnormalities, cardiac diseases, feeding problems and impaired growth. Most patients considered their quality of life high. However, according to the parents’ point of view psychic problems were four times more common in propionic acidemia patients than in healthy controls.
Conclusion
Our data show that the outcome of propionic acidemia is still unfavourable, in spite of improved clinical management. Many patients develop long-term complications affecting different organ systems. Impairment of neurocognitive development is of special concern. Nevertheless, self-assessment of quality of life of the patients and their parents yielded rather positive results.
doi:10.1186/1750-1172-8-6
PMCID: PMC3568723  PMID: 23305374
Propionic acidemia; Branched-chain amino acids; Outcome; Quality of life; Clinical course; Physical development; Neurocognitive development; IQ; Long-term complications; Propionyl-coenzyme A carboxylase deficiency
6.  Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal recognition particle 
Molecular Biology of the Cell  2012;23(3):464-479.
The YidC insertase also integrates multispanning membrane proteins that had been considered to be exclusively SecYEG dependent. Only membrane proteins that require SecA can be inserted only via SecYEG. Targeting to YidC is SRP dependent, and the C-terminus of YidC cross-links to SRP, FtsY, and ribosomal subunits.
Protein insertion into the bacterial inner membrane is facilitated by SecYEG or YidC. Although SecYEG most likely constitutes the major integration site, small membrane proteins have been shown to integrate via YidC. We show that YidC can also integrate multispanning membrane proteins such as mannitol permease or TatC, which had been considered to be exclusively integrated by SecYEG. Only SecA-dependent multispanning membrane proteins strictly require SecYEG for integration, which suggests that SecA can only interact with the SecYEG translocon, but not with the YidC insertase. Targeting of multispanning membrane proteins to YidC is mediated by signal recognition particle (SRP), and we show by site-directed cross-linking that the C-terminus of YidC is in contact with SRP, the SRP receptor, and ribosomal proteins. These findings indicate that SRP recognizes membrane proteins independent of the downstream integration site and that many membrane proteins can probably use either SecYEG or YidC for integration. Because protein synthesis is much slower than protein transport, the use of YidC as an additional integration site for multispanning membrane proteins may prevent a situation in which the majority of SecYEG complexes are occupied by translating ribosomes during cotranslational insertion, impeding the translocation of secretory proteins.
doi:10.1091/mbc.E11-07-0590
PMCID: PMC3268725  PMID: 22160593
7.  Novel Transporter Required for Biogenesis of cbb3-Type Cytochrome c Oxidase in Rhodobacter capsulatus 
mBio  2012;3(1):e00293-11.
ABSTRACT
The acquisition, delivery, and incorporation of metals into their respective metalloproteins are important cellular processes. These processes are tightly controlled in order to prevent exposure of cells to free-metal concentrations that could yield oxidative damage. Copper (Cu) is one such metal that is required as a cofactor in a variety of proteins. However, when present in excessive amounts, Cu is toxic due to its oxidative capability. Cytochrome c oxidases (Coxs) are among the metalloproteins whose assembly and activity require the presence of Cu in their catalytic subunits. In this study, we focused on the acquisition of Cu for incorporation into the heme-Cu binuclear center of the cbb3-type Cox (cbb3-Cox) in the facultative phototroph Rhodobacter capsulatus. Genetic screens identified a cbb3-Cox defective mutant that requires Cu2+ supplementation to produce an active cbb3-Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required for cbb3-Cox biogenesis. In the absence of CcoA, the cellular Cu content decreases and cbb3-Cox assembly and activity become defective. CcoA shows homology to major facilitator superfamily (MFS)-type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein has been implicated in cbb3-Cox biogenesis. These findings provide novel insights into the maturation and assembly of membrane-integral metalloproteins and on a hitherto-unknown function(s) of MFS-type transporters in bacterial Cu acquisition.
IMPORTANCE
Biogenesis of energy-transducing membrane-integral enzymes, like the heme copper-containing cytochrome c oxidases, and the acquisition of transition metals, like copper, as their catalytic cofactors are vital processes for all cells. These widespread and well-controlled processes are poorly understood in all organisms, including bacteria. Defects in these processes lead to severe mitochondrial diseases in humans and poor crop yields in plants. In this study, using the facultative phototroph Rhodobacter capsulatus as a model organism, we report on the discovery of a novel major facilitator superfamily (MFS)-type transporter (CcoA) that affects cellular copper content and cbb3-type cytochrome c oxidase production in bacteria.
doi:10.1128/mBio.00293-11
PMCID: PMC3266609  PMID: 22294680
8.  Signal sequence–independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle 
Molecular Biology of the Cell  2011;22(13):2309-2323.
Our study reveals an alternative route in the SRP-dependent protein targeting pathway that includes a preassembled, membrane-bound SRP-SR complex. This alternative route is fully sufficient to maintain cell viability in the absence of a soluble SRP.
Protein targeting by the signal recognition particle (SRP) and the bacterial SRP receptor FtsY requires a series of closely coordinated steps that monitor the presence of a substrate, the membrane, and a vacant translocon. Although the influence of substrate binding on FtsY-SRP complex formation is well documented, the contribution of the membrane is largely unknown. In the current study, we found that negatively charged phospholipids stimulate FtsY-SRP complex formation. Phospholipids act on a conserved positively charged amphipathic helix in FtsY and induce a conformational change that strongly enhances the FtsY-lipid interaction. This membrane-bound, signal sequence–independent FtsY-SRP complex is able to recruit RNCs to the membrane and to transfer them to the Sec translocon. Significantly, the same results were also observed with an artificial FtsY-SRP fusion protein, which was tethered to the membrane via a transmembrane domain. This indicates that substrate recognition by a soluble SRP is not essential for cotranslational targeting in Escherichia coli. Our findings reveal a remarkable flexibility of SRP-dependent protein targeting, as they indicate that substrate recognition can occur either in the cytosol via ribosome-bound SRP or at the membrane via a preassembled FtsY-SRP complex.
doi:10.1091/mbc.E11-02-0152
PMCID: PMC3128533  PMID: 21551068
9.  The Putative Assembly Factor CcoH Is Stably Associated with the cbb3-Type Cytochrome Oxidase ▿  
Journal of Bacteriology  2010;192(24):6378-6389.
Cytochrome oxidases are perfect model substrates for analyzing the assembly of multisubunit complexes because the need for cofactor incorporation adds an additional level of complexity to their assembly. cbb3-type cytochrome c oxidases (cbb3-Cox) consist of the catalytic subunit CcoN, the membrane-bound c-type cytochrome subunits CcoO and CcoP, and the CcoQ subunit, which is required for cbb3-Cox stability. Biogenesis of cbb3-Cox proceeds via CcoQP and CcoNO subcomplexes, which assemble into the active cbb3-Cox. Most bacteria expressing cbb3-Cox also contain the ccoGHIS genes, which encode putative cbb3-Cox assembly factors. Their exact function, however, has remained unknown. Here we analyzed the role of CcoH in cbb3-Cox assembly and showed that CcoH is a single spanning-membrane protein with an N-terminus-out-C-terminus-in (Nout-Cin) topology. In its absence, neither the fully assembled cbb3-Cox nor the CcoQP or CcoNO subcomplex was detectable. By chemical cross-linking, we demonstrated that CcoH binds primarily via its transmembrane domain to the CcoP subunit of cbb3-Cox. A second hydrophobic stretch, which is located at the C terminus of CcoH, appears not to be required for contacting CcoP, but deleting it prevents the formation of the active cbb3-Cox. This suggests that the second hydrophobic domain is required for merging the CcoNO and CcoPQ subcomplexes into the active cbb3-Cox. Surprisingly, CcoH does not seem to interact only transiently with the cbb3-Cox but appears to stay tightly associated with the active, fully assembled complex. Thus, CcoH behaves more like a bona fide subunit of the cbb3-Cox than an assembly factor per se.
doi:10.1128/JB.00988-10
PMCID: PMC3008534  PMID: 20952576
10.  A reference dataset for verifying numerical electrophysiological heart models 
Background
The evaluation, verification and comparison of different numerical heart models are difficult without a commonly available database that could be utilized as a reference. Our aim was to compile an exemplary dataset.
Methods
The following methods were employed: Magnetic Resonance Imaging (MRI) of heart and torso, Body Surface Potential Maps (BSPM) and MagnetoCardioGraphy (MCG) maps. The latter were recorded simultaneously from the same individuals a few hours after the MRI sessions.
Results
A training dataset is made publicly available; datasets for blind testing will remain undisclosed.
Conclusions
While the MRI data may provide a common input that can be applied to different numerical heart models, the verification and comparison of different models can be performed by comparing the measured biosignals with forward calculated signals from the models.
doi:10.1186/1475-925X-10-11
PMCID: PMC3037925  PMID: 21272330
11.  Strong cooperativity between subunits in voltage-gated proton channels 
Voltage-activated proton (HV) channels are essential components in the innate immune response. HV channels are dimeric proteins with one proton permeation pathway per subunit. It is not known how HV channels are activated by voltage and whether there is any cooperativity between subunits during voltage activation. Using cysteine accessibility measurements and voltage clamp fluorometry, we show data that are consistent with that the fourth transmembrane segment S4 functions as the voltage sensor in HV channels from Ciona intestinalis. Surprisingly, in a dimeric HV channel, S4 in both subunits have to move to activate the two proton permeation pathways. In contrast, if HV subunits are prevented from dimerizing, then the movement of a single S4 is sufficient to activate the proton permeation pathway in a subunit. These results suggest a strong cooperativity between subunits in dimeric HV channels.
doi:10.1038/nsmb.1739
PMCID: PMC2935852  PMID: 20023639
12.  Depletion of the Signal Recognition Particle Receptor Inactivates Ribosomes in Escherichia coli▿  
Journal of Bacteriology  2009;191(22):7017-7026.
The signal recognition particle (SRP)-dependent cotranslational targeting of proteins to the cytoplasmic membrane in bacteria or the endoplasmic reticulum membrane in eukaryotes is an essential process in most living organisms. Eukaryotic cells have been shown to respond to an impairment of the SRP pathway by (i) repressing ribosome biogenesis, resulting in decreased protein synthesis, and (ii) by increasing the expression of protein quality control mechanisms, such as chaperones and proteases. In the current study, we have analyzed how bacteria like Escherichia coli respond to a gradual depletion of FtsY, the bacterial SRP receptor. Our analyses using cell-free transcription/translation systems showed that FtsY depletion inhibits the translation of both SRP-dependent and SRP-independent proteins. This synthesis defect is the result of a multifaceted response that includes the upregulation of the ribosome-inactivating protein ribosome modulation factor (RMF). Although the consequences of these responses in E. coli are very similar to some of the effects also observed in eukaryotic cells, one striking difference is that E. coli obviously does not reduce the rate of protein synthesis by downregulating ribosome biogenesis. Instead, the upregulation of RMF leads to a direct and reversible inhibition of translation.
doi:10.1128/JB.00208-09
PMCID: PMC2772476  PMID: 19749044
13.  Predominant membrane localization is an essential feature of the bacterial signal recognition particle receptor 
BMC Biology  2009;7:76.
Background
The signal recognition particle (SRP) receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs) to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR) is a heterodimeric protein complex, consisting of two unrelated GTPases. The SRβ subunit is an integral membrane protein, which tethers the SRP-interacting SRα subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SRβ subunit and consists of only the SRα homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of Escherichia coli. So far, the exact function of the soluble SR in E. coli is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting.
Results
In the current study we have determined the contribution of soluble FtsY to co-translational targeting in vitro and have re-analysed the localization of FtsY in vivo by fluorescence microscopy. Our data show that FtsY can bind to SRP-ribosome nascent chains (RNCs) in the absence of membranes. However, these soluble FtsY-SRP-RNC complexes are not efficiently targeted to the membrane. In contrast, we observed effective targeting of SRP-RNCs to membrane-bond FtsY. These data show that soluble FtsY does not contribute significantly to cotranslational targeting in E. coli. In agreement with this observation, our in vivo analyses of FtsY localization in bacterial cells by fluorescence microscopy revealed that the vast majority of FtsY was localized to the inner membrane and that soluble FtsY constituted only a negligible species in vivo.
Conclusion
The exact function of the SRP receptor (SR) in bacteria has so far been enigmatic. Our data show that the bacterial SR is almost exclusively membrane-bound in vivo, indicating that the presence of a soluble SR is probably an artefact of cell fractionation. Thus, co-translational targeting in bacteria does not involve the formation of a soluble SR-signal recognition particle (SRP)-ribosome nascent chain (RNC) intermediate but requires membrane contact of FtsY for efficient SRP-RNC recruitment.
doi:10.1186/1741-7007-7-76
PMCID: PMC2780400  PMID: 19912622
14.  Visualization of Distinct Entities of the SecYEG Translocon during Translocation and Integration of Bacterial Proteins 
Molecular Biology of the Cell  2009;20(6):1804-1815.
The universally conserved SecYEG/Sec61 translocon constitutes the major protein-conducting channel in the cytoplasmic membrane of bacteria and the endoplasmic reticulum membrane of eukaryotes. It is engaged in both translocating secretory proteins across the membrane as well as in integrating membrane proteins into the lipid phase of the membrane. In the current study we have detected distinct SecYEG translocon complexes in native Escherichia coli membranes. Blue-Native-PAGE revealed the presence of a 200-kDa SecYEG complex in resting membranes. When the SecA-dependent secretory protein pOmpA was trapped inside the SecYEG channel, a smaller SecY-containing complex of ∼140-kDa was observed, which probably corresponds to a monomeric SecYEG–substrate complex. Trapping the SRP-dependent polytopic membrane protein mannitol permease in the SecYEG translocon, resulted in two complexes of 250 and 600 kDa, each containing both SecY and the translocon-associated membrane protein YidC. The appearance of both complexes was correlated with the number of transmembrane domains that were exposed during targeting of mannitol permease to the membrane. These results suggest that the assembly or the stability of the bacterial SecYEG translocon is influenced by the substrate that needs to be transported.
doi:10.1091/mbc.E08-08-0886
PMCID: PMC2655265  PMID: 19158385
15.  Stability of the cbb3-Type Cytochrome Oxidase Requires Specific CcoQ-CcoP Interactions▿  
Journal of Bacteriology  2008;190(16):5576-5586.
Cytochrome cbb3-type oxidases are members of the heme copper oxidase superfamily and are composed of four subunits. CcoN contains the heme b-CuB binuclear center where oxygen is reduced, while CcoP and CcoO are membrane-bound c-type cytochromes thought to channel electrons from the donor cytochrome into the binuclear center. Like many other bacterial members of this superfamily, the cytochrome cbb3-type oxidase contains a fourth, non-cofactor-containing subunit, which is termed CcoQ. In the present study, we analyzed the role of CcoQ on the stability and activity of Rhodobacter capsulatus cbb3-type oxidase. Our data showed that CcoQ is a single-spanning membrane protein with a Nout-Cin topology. In the absence of CcoQ, cbb3-type oxidase activity is significantly reduced, irrespective of the growth conditions. Blue native polyacrylamide gel electrophoresis analyses revealed that the lack of CcoQ specifically impaired the stable recruitment of CcoP into the cbb3-type oxidase complex. This suggested a specific CcoQ-CcoP interaction, which was confirmed by chemical cross-linking. Collectively, our data demonstrated that in R. capsulatus CcoQ was required for optimal cbb3-type oxidase activity because it stabilized the interaction of CcoP with the CcoNO core complex, leading subsequently to the formation of the active 230-kDa cbb3-type oxidase complex.
doi:10.1128/JB.00534-08
PMCID: PMC2519385  PMID: 18556791
16.  The Glutamate-Activated Anion Conductance in Excitatory Amino Acid Transporters Is Gated Independently by the Individual Subunits 
Excitatory amino acid transporters (EAATs) use sodium and potassium gradients to remove glutamate from the synapse and surrounding extracellular space, thereby sustaining efficient synaptic transmission and maintaining extracellular glutamate concentrations at subneurotoxic levels. In addition to sodium-driven glutamate uptake, EAATs also mediate a glutamate-activated chloride conductance via a channel-like mechanism. EAATs are trimeric proteins and are thought to comprise three identical subunits. Previous studies have shown that the sodium-driven uptake of glutamate occurs independently in each of the three subunits. In contrast, a recent study reports high Hill coefficients for the activation of EAAT anion currents by glutamate and suggests that the subunits function cooperatively in gating the chloride conductance. In the present work, we find that the Hill coefficient for the activation of the anion current by glutamate is ~1 in both EAAT3 and EAAT4. Furthermore, we also used fluorescent labeling and inactivation correlation on EAAT3 and EAAT4 to determine whether the glutamate-activated chloride conductance is gated independently or cooperatively by the transporters. We found that both glutamate uptake currents and glutamate-activated chloride currents are mediated independently by each subunit of an EAAT multimer. It has been suggested that EAAT subtypes with particularly large anion conductances can directly influence the excitability of presynaptic terminals in certain neurons. Thus, the finding that the anion conductance is gated independently, rather than cooperatively, is important because it significantly alters predictions of the influence that EAAT-mediated anion currents will have on synaptic transmission at low glutamate concentrations.
doi:10.1523/JNEUROSCI.0118-07.2007
PMCID: PMC2435202  PMID: 17360917
anion conductance; chloride; independence; cooperativity; FLIC; glutamate transporter
17.  Membrane binding of the bacterial signal recognition particle receptor involves two distinct binding sites 
The Journal of Cell Biology  2006;174(5):715-724.
Cotranslational protein targeting in bacteria is mediated by the signal recognition particle (SRP) and FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRα subunit of eukaryotes, which is tethered to the membrane via its interaction with the membrane-integral SRβ subunit. Despite the lack of a membrane-anchoring subunit, 30% of FtsY in Escherichia coli are found stably associated with the cytoplasmic membrane. However, the mechanisms that are involved in this membrane association are only poorly understood. Our data indicate that membrane association of FtsY involves two distinct binding sites and that binding to both sites is stabilized by blocking its GTPase activity. Binding to the first site requires only the NG-domain of FtsY and confers protease protection to FtsY. Importantly, the SecY translocon provides the second binding site, to which FtsY binds to form a carbonate-resistant 400-kD FtsY–SecY translocon complex. This interaction is stabilized by the N-terminal A-domain of FtsY, which probably serves as a transient lipid anchor.
doi:10.1083/jcb.200606093
PMCID: PMC2064314  PMID: 16923832
18.  Pseudo current density maps of electrophysiological heart, nerve or brain function and their physical basis 
Background
In recent years the visualization of biomagnetic measurement data by so-called pseudo current density maps or Hosaka-Cohen (HC) transformations became popular.
Methods
The physical basis of these intuitive maps is clarified by means of analytically solvable problems.
Results
Examples in magnetocardiography, magnetoencephalography and magnetoneurography demonstrate the usefulness of this method.
Conclusion
Hardware realizations of the HC-transformation and some similar transformations are discussed which could advantageously support cross-platform comparability of biomagnetic measurements.
doi:10.1186/1477-044X-4-5
PMCID: PMC1660567  PMID: 17040559
19.  Ligand crowding at a nascent signal sequence 
The Journal of Cell Biology  2003;163(1):35-44.
We have systematically analyzed the molecular environment of the signal sequence of a growing secretory protein from Escherichia coli using a stage- and site-specific cross-linking approach. Immediately after emerging from the ribosome, the signal sequence of pOmpA is accessible to Ffh, the protein component of the bacterial signal recognition particle, and to SecA, but it remains attached to the surface of the ribosome via protein L23. These contacts are lost upon further growth of the nascent chain, which brings the signal sequence into sole proximity to the chaperone Trigger factor (TF). In its absence, nascent pOmpA shows extended contacts with L23, and even long chains interact in these conditions proficiently with Ffh. Our results suggest that upon emergence from the ribosome, the signal sequence of an E. coli secretory protein gradually becomes sequestered by TF. Although TF thereby might control the accessibility of pOmpA's signal sequence to Ffh and SecA, it does not influence interaction of pOmpA with SecB.
doi:10.1083/jcb.200306069
PMCID: PMC2173441  PMID: 14530384
L23; nascent polypeptide chains; protein targeting; signal recognition particle; Trigger factor
20.  Population Dynamics of Persistent Staphylococcus aureus Isolated from the Airways of Cystic Fibrosis Patients during a 6-Year Prospective Study 
Journal of Clinical Microbiology  2003;41(9):4424-4427.
Molecular typing of normal (n = 456) and small-colony-variant (SCV; n = 239) Staphylococcus aureus isolates cultured from the airways of 52 of 72 cystic fibrosis (CF) patients (72.2%) during a 6-year prospective study revealed a median long-term persistence of 37 months (range, 6 to 70). SCV persisted longer in the airways than the normal S. aureus (statistically not significant). Pulsed-field gel electrophoresis identified six prevalent clonal lineages, which were cultured from more than one patient (3 to 12 patients), and 39 individual clones, which were isolated only from single patients. The SCV phenotype was not restricted to a distinct clonal lineage but occurred in many different clones. Most patients (33 of 52, 63.46%) harbored single clones. This study provides a basis for improved understanding of S. aureus colonization and infection dynamics in CF patients.
doi:10.1128/JCM.41.9.4424-4427.2003
PMCID: PMC193812  PMID: 12958283
21.  Dissecting the Translocase and Integrase Functions of the Escherichia coli Secyeg Translocon 
The Journal of Cell Biology  2000;150(3):689-694.
Recent evidence suggests that in Escherichia coli, SecA/SecB and signal recognition particle (SRP) are constituents of two different pathways targeting secretory and inner membrane proteins to the SecYEG translocon of the plasma membrane. We now show that a secY mutation, which compromises a functional SecY–SecA interaction, does not impair the SRP-mediated integration of polytopic inner membrane proteins. Furthermore, under conditions in which the translocation of secretory proteins is strictly dependent on SecG for assisting SecA, the absence of SecG still allows polytopic membrane proteins to integrate at the wild-type level. These results indicate that SRP-dependent integration and SecA/SecB-mediated translocation do not only represent two independent protein delivery systems, but also remain mechanistically distinct processes even at the level of the membrane where they engage different domains of SecY and different components of the translocon. In addition, the experimental setup used here enabled us to demonstrate that SRP-dependent integration of a multispanning protein into membrane vesicles leads to a biologically active enzyme.
PMCID: PMC2175189  PMID: 10931878
membrane proteins; protein transport signal recognition particle; SecA; SecYEG complex
22.  Isolation and Characterization of Rhodobacter capsulatus Mutants Affected in Cytochrome cbb3 Oxidase Activity 
Journal of Bacteriology  1998;180(4):969-978.
The facultative phototrophic bacterium Rhodobacter capsulatus contains only one form of cytochrome (cyt) c oxidase, which has recently been identified as a cbb3-type cyt c oxidase. This is unlike other related species, such as Rhodobacter sphaeroides and Paracoccus denitrificans, which contain an additional mitochondrial-like aa3-type cyt c oxidase. An extensive search for mutants affected in cyt c oxidase activity in R. capsulatus led to the isolation of at least five classes of mutants. Plasmids complementing them to a wild-type phenotype were obtained for all but one of these classes from a chromosomal DNA library. The first class of mutants contained mutations within the structural genes (ccoNOQP) of the cyt cbb3 oxidase. Sequence analysis of these mutants and of the plasmids complementing them revealed that ccoNOQP in R. capsulatus is not flanked by the oxygen response regulator fnr, which is located upstream of these genes in other species. Genetic and biochemical characterizations of mutants belonging to this group indicated that the subunits CcoN, CcoO, and CcoP are required for the presence of an active cyt cbb3 oxidase, and unlike in Bradyrhizobium japonicum, no active CcoN-CcoO subcomplex was found in R. capsulatus. In addition, mutagenesis experiments indicated that the highly conserved open reading frame 277 located adjacent to ccoNOQP is required neither for cyt cbb3 oxidase activity or assembly nor for respiratory or photosynthetic energy transduction in R. capsulatus. The remaining cyt c oxidase-minus mutants mapped outside of ccoNOQP and formed four additional groups. In one of these groups, a fully assembled but inactive cyt cbb3 oxidase was found, while another group had only extremely small amounts of it. The next group was characterized by a pleiotropic effect on all membrane-bound c-type cytochromes, and the remaining mutants not complemented by the plasmids complementing the first four groups formed at least one additional group affecting the biogenesis of the cyt cbb3 oxidase of R. capsulatus.
PMCID: PMC106979  PMID: 9473054
23.  Diversity of Cystathionine β-Synthase Haplotypes Bearing the Most Common Homocystinuria Mutation c.833T>C: A Possible Role for Gene Conversion 
Human Mutation  2006;28(3):255-264.
Homozygosity or compound heterozygosity for the c.833T>C transition (p.I278T) in the cystathionine beta-synthase (CBS) gene represents the most common cause of pyridoxine-responsive homocystinuria in Western Eurasians. However, the frequency of the pathogenic c.833C allele, as observed in healthy newborns from several European countries (qc.833C ≊ 3.3 × 10–3), is ∼20-fold higher than expected on the basis of the observed number of symptomatic homocystinuria patients carrying this mutation (qc.833C ≊ 0.18 × 10–3), implying clinical underascertainment. Intriguingly, the c.833C mutation is also present in combination with a 68-bp insertion, c.[833C; 844_845ins68], in a substantial proportion of chromosomes from nonhomocystinuric individuals worldwide. We have sought to study the relationship between the pathogenic and nonpathogenic c.833C-bearing chromosomes and to determine whether the pathogenic c.[833C; −] chromosomes are identical-by-descent or instead arose by recurrent mutation. Initial haplotype analysis of 780 randomly selected Czech and sub-Saharan African wild-type chromosomes, employing 12 intragenic markers, revealed 29 distinct CBS haplotypes, of which 10 carried the c.[833C; 844_845ins68] combination; none carried an isolated c.833C or c.844_845ins68 mutation. Subsequent examination of 69 pathogenic c.[833C; −] chromosomes, derived from homocystinuria patients of predominantly European origin, disclosed three unrelated haplotypes that differed from their wild-type counterparts by virtue of the presence of c.833C, thereby indicating that c.833T>C transition has occurred repeatedly and independently in the past. Since c.833T does not reside within an obvious mutational hotspot, we surmise that the three pathogenic and comparatively prevalent c.[833C; −] chromosomes may have originated by recurrent gene conversion employing the common nonpathogenic c.[833C; 844_845ins68] chromosomes as templates. Hum Mutat 28(3), 255–264, 2007. Published 2006 Wiley-Liss, Inc.†
doi:10.1002/humu.20430
PMCID: PMC2630376  PMID: 17072863
homocysteine; homocystinuria; haplotype; pyridoxal 5′phosphate; cystathionine beta-synthase; CBS; gene conversion
24.  In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways of Escherichia coli 
Molecular Biology of the Cell  1999;10(7):2163-2173.
The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coli which, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and ΔμH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.
PMCID: PMC25430  PMID: 10397756

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