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Subunit C (subunit + c)
Selected AbstractsStructural evidence for a constant c11 ring stoichiometry in the sodium F-ATP synthaseFEBS JOURNAL, Issue 21 2005Thomas Meier The Na+ -dependent F-ATP synthases of Ilyobacter tartaricus and Propionigenium modestum contain membrane-embedded ring-shaped c subunit assemblies with a stoichiometry of 11. Subunit c from either organism was overexpressed in Escherichia coli using a plasmid containing the corresponding gene, extracted from the membrane using detergent and then purified. Subsequent analyses by SDS/PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the majority migrated as monomers. The population of rings consisted mainly of c11, but more slowly migrating assemblies were also found, which might reflect other c ring stoichiometries. We show that they consisted of higher aggregates of homogeneous c11 rings and/or assemblies of c11 rings and single c monomers. Atomic force microscopy topographs of c rings reconstituted into lipid bilayers showed that the c ring assemblies had identical diameters and that stoichiometries throughout all rings resolved at high resolution. This finding did not depend on whether the rings were assembled into crystalline or densely packed assemblies. Most of these rings represented completely assembled undecameric complexes. Occasionally, rings lacking a few subunits or hosting additional subunits in their cavity were observed. The latter rings may represent the aggregates between c11 and c1, as observed by SDS/PAGE. Our results are congruent with a stable c11 ring stoichiometry that seems to not be influenced by the expression level of subunit c in the bacteria. [source] Molecular and physiological analysis of an OxyR-regulated ahpC promoter in Xanthomonas campestris pv. phaseoliMOLECULAR MICROBIOLOGY, Issue 6 2000Suvit Loprasert In Xanthomonas campestris pv. phaseoli, a gene for the alkyl hydroperoxide reductase subunit C (ahpC) had unique patterns of regulation by various forms of OxyR. Reduced OxyR repressed expression of the gene, whereas oxidized OxyR activated its expression. This dual regulation of ahpC is unique and unlike all other OxyR-regulated genes. The ahpC transcription start site was determined. Analysis of the region upstream of the site revealed promoter sequences that had high homology to the Xanthomonas consensus promoter sequence. Data from gel shift experiments indicated that both reduced and oxidized OxyR could bind to the ahpC regulatory region. Moreover, the reduced and the oxidized forms of OxyR gave different DNase I footprint patterns, indicating that they bound to different sites. The oxidized OxyR binding site overlapped the ,35 region of the ahpC promoter by a few bases. This position is consistent with the role of the protein in activating transcription of the gene. Binding of reduced OxyR to the ahpC promoter showed an extended DNase I footprint and DNase I hypersensitive sites, suggesting that binding of the protein caused a shift in the binding site and bending of the target DNA. In addition, binding of reduced OxyR completely blocked the ,35 region of the ahpC promoter and prevented binding of RNA polymerase, leading to repression of the gene. Monitoring of the ahpC promoter activity in vivo confirmed the location of the oxidized OxyR binding site required for activation of the promoter. A mutant that separated OxyR regulation from basal ahpC promoter activity was constructed. The mutant was unable to respond to oxidants by increasing ahpC expression. Physiologically, it had a slower aerobic growth rate and was more sensitive to organic peroxide killing. This indicated that oxidant induction of ahpC has important physiological roles in normal growth and during oxidative stress. [source] V-ATPase deactivation in blowfly salivary glands is mediated by protein phosphatase 2CARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009Martin Voss Abstract The activity of vacuolar H+ -ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT). 5-HT induces, via protein kinase A, the phosphorylation of V-ATPase subunit C and the assembly of V-ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V-ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK-506) do not prevent V-ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg2+ level caused by loading secretory cells with EDTA-AM leads to the activation of proton pumping in the absence of 5-HT, prolongs the 5-HT-induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V-ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg2+, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc. [source] Structural comparison of Escherichia colil -asparaginase in two monoclinic space groupsACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2003Mario Sanches The functional l -asparaginase from Escherichia coli is a homotetramer with a molecular weight of about 142,kDa. The X-ray structure of the enzyme, crystallized in a new form (space group C2) and refined to 1.95,Å resolution, is compared with that of the previously determined crystal form (space group P21). The asymmetric unit of the new crystal form contains an l -asparaginase dimer instead of the tetramer found in the previous crystal form. It is found that crystal contacts practically do not affect the conformation of the protein. It is shown that subunit C of the tetrameric form is in a conformation which is systematically different from that of all other subunits in both crystal forms. Major conformational differences are confined to the lid loop (residues 14,27). In addition, the stability of this globular protein is analyzed in terms of the interactions between hydrophobic parts of the subunits. [source] Neuron-specific expression of atp6v0c2 in zebrafish CNSDEVELOPMENTAL DYNAMICS, Issue 9 2010Ah-Young Chung Abstract Vacuolar ATPase (V-ATPase) is a multi-subunit enzyme that plays an important role in the acidification of a variety of intracellular compartments. ATP6V0C is subunit c of the V0 domain that forms the proteolipid pore of the enzyme. In the present study, we investigated the neuron-specific expression of atp6v0c2, a novel isoform of the V-ATPase c-subunit, during the development of the zebrafish CNS. Zebrafish atp6v0c2 was isolated from a genome-wide analysis of the zebrafish mibta52b mutant designed to identify genes differentially regulated by Notch signaling. Whole-mount in situ hybridization revealed that atp6v0c2 is expressed in a subset of CNS neurons beginning several hours after the emergence of post-mitotic neurons. The ATP6V0C2 protein is co-localized with the presynaptic vesicle marker, SV2, suggesting that it is involved in neurotransmitter storage and/or secretion in neurons. In addition, the loss-of-function experiment suggests that ATP6V0C2 is involved in the control of neuronal excitability. Developmental Dynamics 239:2501,2508, 2010. © 2010 Wiley-Liss, Inc. [source] Structural evidence for a constant c11 ring stoichiometry in the sodium F-ATP synthaseFEBS JOURNAL, Issue 21 2005Thomas Meier The Na+ -dependent F-ATP synthases of Ilyobacter tartaricus and Propionigenium modestum contain membrane-embedded ring-shaped c subunit assemblies with a stoichiometry of 11. Subunit c from either organism was overexpressed in Escherichia coli using a plasmid containing the corresponding gene, extracted from the membrane using detergent and then purified. Subsequent analyses by SDS/PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the majority migrated as monomers. The population of rings consisted mainly of c11, but more slowly migrating assemblies were also found, which might reflect other c ring stoichiometries. We show that they consisted of higher aggregates of homogeneous c11 rings and/or assemblies of c11 rings and single c monomers. Atomic force microscopy topographs of c rings reconstituted into lipid bilayers showed that the c ring assemblies had identical diameters and that stoichiometries throughout all rings resolved at high resolution. This finding did not depend on whether the rings were assembled into crystalline or densely packed assemblies. Most of these rings represented completely assembled undecameric complexes. Occasionally, rings lacking a few subunits or hosting additional subunits in their cavity were observed. The latter rings may represent the aggregates between c11 and c1, as observed by SDS/PAGE. Our results are congruent with a stable c11 ring stoichiometry that seems to not be influenced by the expression level of subunit c in the bacteria. [source] NMR investigations of subunit c of the ATP synthase from Propionigenium modestum in chloroform/methanol/water (4 : 4 : 1)FEBS JOURNAL, Issue 7 2002Ulrich Matthey The subunit c from the ATP synthase of Propionigenium modestum was studied by NMR in chloroform/methanol/water (4 : 4 : 1). In this solvent, subunit c consists of two helical segments, comprised of residues L5 to I26 and G29 to N82, respectively. On comparing the secondary structure of subunit c from P. modestum in the organic solvent mixture with that in dodecylsulfate micelles several deviations became apparent: in the organic solvent, the interruption of the ,,helical structure within the conserved GXGXGXGX motif was shortened from five to two residues, the prominent interruption of the ,,helical structure in the cystoplasmic loop region was not apparent, and neither was there a break in the ,,helix after the sodium ion-binding Glu65 residue. The folding of subunit c of P. modestum in the organic solvent also deviated from that of Escherichia coli in the same environment, the most important difference being that subunit c of P. modestum did not adopt a stable hairpin structure like subunit c of E. coli. [source] Identification of novel splice variants of the human catalytic subunit c, of cAMP-dependent protein kinaseFEBS JOURNAL, Issue 19 2001Sigurd Ørstavik Four different isoforms of the catalytic subunit of cAMP-dependent protein kinase, termed C,, C,, C, and PrKX have been identified. Here we demonstrate that the human C, gene encodes six splice variants, designated C,1, C,2, C,3, C,4, C,4ab and C,4abc. The C, splice variants differ in their N-terminal ends due to differential splicing of four different forms of exon 1 designated exon 1-1, 1-2, 1-3, 1-4 and three exons designated a, b and c. All these exons are located upstream of exon 2 in the C, gene. The previously identified human C, variant has been termed C,1, and is similar to the C, isoform identified in the mouse, ox, pig and several other mammals. Human C,2, which is the homologue of bovine C,2, has no homologue in the mouse. Human C,3 and C,4 are homologous to the murine C,3 and C,2 splice variants, whereas human C,4ab and C,4abc represent novel isofoms previously not identified in any other species. At the mRNA level, the C, splice variants reveal tissue specific expression. C,1 was most abundantly expressed in the brain, with low-level expression in several other tissues. The C,3 and C,4 splice variants were uniquely expressed in human brain in contrast to C,2, which was most abundantly expressed in tissues of the immune system, with no detectable expression in brain. We suggest that the various C, splice variants when complexed with regulatory subunits may give rise to novel holoenzymes of protein kinase A that may be important for mediating specific effects of cAMP. [source] Distal intestinal gene expression in Atlantic salmon (Salmo salar L.) fed genetically modified maizeAQUACULTURE NUTRITION, Issue 1 2009M.K. FRØYSTAD-SAUGEN Abstract In the current experiment, RNA was isolated from the distal intestine (DI) of Atlantic salmon-fed fishmeal-based diets containing either genetically modified (GM) maize (Bt maize, Mon810®, Monsanto Company, St. Louis, Missouri, USA) or its conventional near-isogenic parental line (non-GM) for 82 days, both at 300 g kg,1 inclusion. From a suppression subtractive hybridization (SSH) cDNA library, 192 clones with similarity to both known and novel Atlantic salmon sequences were identified. Real-time PCR was used to study the differential expression of 10 clones between the dietary groups. Expression of a clone showing high protein similarity to proton-dependent high-affinity oligopeptide transporter was significantly upregulated in fish-fed GM maize compared with fish-fed non-GM maize. No significant differences in expression were observed for the nine other clones showing similarity to the following proteins: heat shock protein 90B, procathepsin B, interferon gamma-inducible protein 30, ferritin heavy subunit, serum lectin isoform/C-type mannose-binding lectin, fatty acid-binding protein/gastrotropin, ATP synthase [H+ transporting, mitochondrial F0 complex, subunit c (ATPSYNT)], sonic hedgehog and translationally controlled tumour protein. In conclusion, only minor differences in DI transcriptional gene expression was observed between fish fed the GM and non-GM maize diets. [source] Autosomal Dominant Adult Neuronal Ceroid Lipofuscinosis: a Novel Form of NCL with Granular Osmiophilic Deposits without Palmitoyl Protein Thioesterase 1 DeficiencyBRAIN PATHOLOGY, Issue 4 2003Peter C. G. Nijssen We describe the neuropathological and biochemical autopsy findings in 3 patients with autosomal dominant adult neuronal ceroid lipofuscinosis (ANCL, Parry type; MIM 162350), from a family with 6 affected individuals in 3 generations. Throughout the brain of these patients, there was abundant intraneuronal lysosomal storage of autofluorescent lipopigment granules. Striking loss of neurons in the substantia nigra was found. In contrast, little neuronal cell loss occurred in other cerebral areas, despite massive neuronal inclusions. Visceral storage was present in gut, liver, cardiomyocytes, skeletal muscle, and in the skin eccrine glands. The storage material showed highly variable immunoreactivity with antiserum against subunit c of mitochondrial ATP synthase, but uniform strong immunoreactivity for saposin D (sphingolipid activating protein D). Protein electrophoresis of isolated storage material revealed a major protein band of about 14 kDa, recognized in Western blotting by saposin D antiserum (but not subunit c of mitochondrial ATPase (SCMAS) antiserum). Electron microscopy showed ample intraneuronal granular osmiophilic deposits (GRODs), as occurs in CLN1 and congenital ovine NCL. These forms of NCL are caused by the deficiencies of palmitoyl protein thioesterase 1 and cathepsin D, respectively. However, activities of these enzymes were within normal range in our patients. Thus we propose that a gene distinct from the cathepsin D and CLN1-CLN8 genes is responsible for this autosomal dominant form of ANCL. [source] | |||||||||||||