Human granulocytic ehrlichiosis agent and Ehrlichia chaffeensis reside in different cytoplasmic compartments in HL-60 cells.
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The human granulocytic ehrlichiosis (HGE) agent resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. Double immunofluorescence labeling was used to characterize the nature of the HGE agent replicative inclusions and to compare them with inclusions containing the human monocytic ehrlichia, Ehrlichia chaffeensis, in HL-60 cells. Although both Ehrlichia spp. can coinfect HL-60 cells, they resided in separate inclusions. Inclusions of both Ehrlichia spp. were not labeled with either anti-lysosome-associated membrane protein 1 or anti-CD63. Accumulation of myeloperoxidase-positive granules were seen around HGE agent inclusions but not around E. chaffeensis inclusions. 3-(2, 4-Dinitroanilino)-3'-amino-N-methyldipropylamine and acridine orange were not localized to either inclusion type. Vacuolar-type H+-ATPase was not colocalized with HGE agent inclusions but was weakly colocalized with E. chaffeensis inclusions. E. chaffeensis inclusions were labeled with the transferrin receptor, early endosomal antigen 1, and rab5, but HGE agent inclusions were not. Some HGE agent and E. chaffeensis inclusions colocalized with major histocompatibility complex class I and II antigens. These two inclusions were not labeled for annexins I, II, IV, and VI; alpha-adaptin; clathrin heavy chain; or beta-coatomer protein. Vesicle-associated membrane protein 2 colocalized to both inclusions. The cation-independent mannose 6-phosphate receptor was not colocalized with either inclusion type. Endogenously synthesized sphingomyelin, from C6-NBD-ceramide, was not incorporated into either inclusion type. Brefeldin A did not affect the growth of either Ehrlichia sp. in HL-60 cells. These results suggest that the HGE agent resides in inclusions which are neither early nor late endosomes and does not fuse with lysosomes or Golgi-derived vesicles, while E. chaffeensis resides in an early endosomal compartment which accumulates the transferrin receptor. (+info)
Cross-linking of two beta subunits in the closed conformation in F1-ATPase.
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In the crystal structure of mitochondrial F1-ATPase, two beta subunits with a bound Mg-nucleotide are in "closed" conformations, whereas the third beta subunit without bound nucleotide is in an "open" conformation. In this "CCO" (beta-closed beta-closed beta-open) conformational state, Ile-390s of the two closed beta subunits, even though they are separated by an intervening alpha subunit, have a direct contact. We replaced the equivalent Ile of the alpha3beta3gamma subcomplex of thermophilic F1-ATPase with Cys and observed the formation of the beta-beta cross-link through a disulfide bond. The analysis of conditions required for the cross-link formation indicates that: (i) F1-ATPase takes the CCO conformation when two catalytic sites are filled with Mg-nucleotide, (ii) intermediate(s) with the CCO conformation are generated during catalytic cycle, (iii) the Mg-ADP inhibited form is in the CCO conformation, and (iv) F1-ATPase dwells in conformational state(s) other than CCO when only one (or none) of catalytic sites is filled by Mg-nucleotide or when catalytic sites are filled by Mg2+-free nucleotide. The alpha3beta3gamma subcomplex containing the beta-beta cross-link retained the activity of uni-site catalysis but lost that of multiple catalytic turnover, suggesting that open-closed transition of beta subunits is required for the rotation of gamma subunit but not for hydrolysis of a single ATP. (+info)
Binding of the transition state analog MgADP-fluoroaluminate to F1-ATPase.
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Escherichia coli F1-ATPase from mutant betaY331W was potently inhibited by fluoroaluminate plus MgADP but not by MgADP alone. beta-Trp-331 fluorescence was used to measure MgADP binding to catalytic sites. Fluoroaluminate induced a very large increase in MgADP binding affinity at catalytic site one, a smaller increase at site two, and no effect at site three. Mutation of either of the critical catalytic site residues beta-Lys-155 or beta-Glu-181 to Gln abolished the effects of fluoroaluminate on MgADP binding. The results indicate that the MgADP-fluoroaluminate complex is a transition state analog and independently demonstrate that residues beta-Lys-155 and (particularly) beta-Glu-181 are important for generation and stabilization of the catalytic transition state. Dicyclohexylcarbodiimide-inhibited enzyme, with 1% residual steady-state ATPase, showed normal transition state formation as judged by fluoroaluminate-induced MgADP binding affinity changes, consistent with a proposed mechanism by which dicyclohexylcarbodiimide prevents a conformational interaction between catalytic sites but does not affect the catalytic step per se. The fluorescence technique should prove valuable for future transition state studies of F1-ATPase. (+info)
EPR spectroscopy of VO2+-ATP bound to catalytic site 3 of chloroplast F1-ATPase from Chlamydomonas reveals changes in metal ligation resulting from mutations to the phosphate-binding loop threonine (betaT168).
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Site-directed mutations were made to the phosphate-binding loop threonine in the beta-subunit of the chloroplast F1-ATPase in Chlamydomonas (betaT168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from betaT168D, betaT168C, and betaT168L mutants had <10% of the wild type rates, as did rates of Mg2+-ATPase activity of purified chloroplast F1-ATPase (CF1). The EPR spectra of VO2+-ATP bound to Site 3 of CF1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF1 from each mutant in both signal intensity and in 51V hyperfine parameters that depend on the equatorial VO2+ ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphate-binding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg2+-ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF1 with entrapped Mg2+-ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand. (+info)
Single copies of subunits d, oligomycin-sensitivity conferring protein, and b are present in the Saccharomyces cerevisiae mitochondrial ATP synthase.
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In the mitochondrial ATP synthase (mtATPase) of the yeast Saccharomyces cerevisiae, the stoichiometry of subunits d, oligomycin-sensitivity conferring protein (OSCP), and b is poorly defined. We have investigated the stoichiometry of these subunits by the application of hexahistidine affinity purification technology. We have previously demonstrated that intact mtATPase complexes incorporating a Hex6-tagged subunit can be isolated via Ni2+-nitrilotriacetic acid affinity chromatography (Bateson, M., Devenish, R. J., Nagley, P., and Prescott, M. (1996) Anal. Biochem. 238, 14-18). Strains were constructed in which Hex6-tagged versions of subunits d, OSCP, and b were coexpressed with the corresponding wild-type subunit. This coexpression resulted in a mixed population of mtATPase complexes containing untagged wild-type and Hex6-tagged subunits. The stoichiometry of each subunit was then assessed by determining whether or not the untagged wild-type subunit could be recovered from Ni2+-nitrilotriacetic acid purifications as an integral component of those complexes absorbed by virtue of the Hex6-tagged subunit. As only the Hex6-tagged subunit was recovered from such purifications, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeast is 1 in each case. (+info)
Effect of acidification on the location of H+-ATPase in cultured inner medullary collecting duct cells.
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In previous studies, our laboratory has utilized a cell line derived from the rat inner medullary collecting duct (IMCD) as a model system for mammalian renal epithelial cell acid secretion. We have provided evidence, from a physiological perspective, that acute cellular acidification stimulates apical exocytosis and elicits a rapid increase in proton secretion that is mediated by an H+-ATPase. The purpose of these experiments was to examine the effect of acute cellular acidification on the distribution of the vacuolar H+-ATPase in IMCD cells in vitro. We utilized the 31-kDa subunit of the H+-ATPase as a marker of the complete enzyme. The distribution of this subunit of the H+-ATPase was evaluated by immunohistochemical techniques (confocal and electron microscopy), and we found that there is a redistribution of these pumps from vesicles to the apical membrane. Immunoblot evaluation of isolated apical membrane revealed a 237 +/- 34% (P < 0.05, n = 9) increase in the 31-kDa subunit present in the membrane fraction 20 min after the induction of cellular acidification. Thus our results demonstrate the presence of this pump subunit in the IMCD cell line in vitro and that cell acidification regulates the shuttling of cytosolic vesicles containing the 31-kDa subunit into the apical membrane. (+info)
Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress.
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The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions. (+info)
Receptor-mediated targeting of fluorescent probes in living cells.
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A strategy was developed to label specified sites in living cells with a wide selection of fluorescent or other probes and applied to study pH regulation in Golgi. cDNA transfection was used to target a single-chain antibody to a specified site such as an organelle lumen. The targeted antibody functioned as a high affinity receptor to trap cell-permeable hapten-fluorophore conjugates. Synthesized conjugates of a hapten (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, phOx) and fluorescent probes (Bodipy Fl, tetramethylrhodamine, fluorescein) were bound with high affinity (approximately 5 nM) and specific localization to the single-chain antibody expressed in the endoplasmic reticulum, Golgi, and plasma membrane of living Chinese hamster ovary cells. Using the pH-sensitive phOx-fluorescein conjugate and ratio imaging microscopy, pH was measured in the lumen of Golgi (pH 6.25 +/- 0.06). Measurements of pH-dependent vacuolar H+/ATPase pump activity and H+ leak in Golgi provided direct evidence that resting Golgi pH is determined by balanced leak-pump kinetics rather than the inability of the H+/ATPase to pump against an electrochemical gradient. Like expression of the green fluorescent protein, the receptor-mediated fluorophore targeting approach permits specific intracellular fluorescence labeling. A significant advantage of the new approach is the ability to target chemical probes with custom-designed spectral and indicator properties. (+info)