Expression of Batis maritima methyl chloride transferase in Escherichia coli

Methyl chloride transferase, a novel enzyme found in several fungi, marine algae, and halophytic plants, is a biological catalyst responsible for the production of atmospheric methyl chloride. A previous paper reports the purification of this methylase from Batis maritima and the isolation of a cDNA clone of the gene for this enzyme. In this paper, we describe the isolation of a genomic clone of the methylase gene and the expression of recombinant methyl chloride transferase in Escherichia coli and compare the kinetic behavior of the wild-type and recombinant enzyme. The recombinant enzyme is active and promotes the production of methyl chloride by E. coli under in vivo conditions. The kinetic data indicate that the recombinant and wild-type enzymes have similar halide (Cl−, Br−, and I−)-binding capacities. Both the recombinant and wild-type enzymes were found to function well in high NaCl concentrations. This high salt tolerance resembles the activity of halobacterial enzymes rather than halophytic plant enzymes. These findings support the hypothesis that this enzyme functions in the control and regulation of the internal concentration of chloride ions in halophytic plant cells.

Induction of epithelial chloride secretion by channel-forming cryptdins 2 and 3

Salt and water secretion from intestinal epithelia requires enhancement of anion permeability across the apical membrane of Cl− secreting cells lining the crypt, the secretory gland of the intestine. Paneth cells located at the base of the small intestinal crypt release enteric defensins (cryptdins) apically into the lumen. Because cryptdins are homologs of molecules known to form anion conductive pores in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes of intestinal Cl− secreting epithelial cells in culture. Of the six peptides tested, cryptdins 2 and 3 stimulated Cl− secretion from polarized monolayers of human intestinal T84 cells. The response was reversible and dose dependent. In contrast, cryptdins 1, 4, 5, and 6 lacked this activity, demonstrating that Paneth cell defensins with very similar primary structures may exhibit a high degree of specificity in their capacity to elicit Cl− secretion. The secretory response was not inhibited by pretreatment with 8-phenyltheophyline (1 μM), or dependent on a concomitant rise in intracellular cAMP or cGMP, indicating that the apically located adenosine and guanylin receptors were not involved. On the other hand, cryptdin 3 elicited a secretory response that correlated with the establishment of an apically located anion conductive channel permeable to carboxyfluorescein. Thus cryptdins 2 and 3 can selectively permeabilize the apical cell membrane of epithelial cells in culture to elicit a physiologic Cl− secretory response. These data define the capability of cryptdins 2 and 3 to function as novel intestinal secretagogues, and suggest a previously undescribed mechanism of paracrine signaling that in vivo may involve the reversible formation of ion conductive channels by peptides released into the crypt microenvironment.

Wheat cytosolic acetyl-CoA carboxylase complements an ACC1 null mutation in yeast

Spores harboring an ACC1 deletion derived from a diploid Saccharomyces cerevisiae strain, in which one copy of the entire ACC1 gene is replaced with a LEU2 cassette, fail to grow. A chimeric gene consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat cytosolic acetyl-CoA carboxylase (ACCase) cDNA, and yeast ACC1 3′ tail was used to complement a yeast ACC1 mutation. The complementation demonstrates that active wheat ACCase can be produced in yeast. At low concentrations of galactose, the activity of the “wheat gene” driven by the GAL10 promoter is low and ACCase becomes limiting for growth, a condition expected to enhance transgenic yeast sensitivity to wheat ACCase-specific inhibitors. An aryloxyphenoxypropionate and two cyclohexanediones do not inhibit growth of haploid yeast strains containing the yeast ACC1 gene, but one cyclohexanedione inhibits growth of the gene-replacement strains at concentrations below 0.2 mM. In vitro, the activity of wheat cytosolic ACCase produced by the gene-replacement yeast strain is inhibited by haloxyfop and cethoxydim at concentrations above 0.02 mM. The activity of yeast ACCase is less affected. The wheat plastid ACCase in wheat germ extract is inhibited by all three herbicides at concentrations below 0.02 mM. Yeast gene-replacement strains will provide a convenient system for the study of plant ACCases.

Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase

Fatty acid synthesis in chloroplasts is regulated by light. The synthesis of malonyl-CoA, which is catalyzed by acetyl-CoA carboxylase (ACCase) and is the first committed step, is modulated by light/dark. Plants have ACCase in plastids and the cytosol. To determine the possible involvement of a redox cascade in light/dark modulation of ACCase, the effect of DTT, a known reductant of S-S bonds, was examined in vitro for the partially purified ACCase from pea plant. Only the plastidic ACCase was activated by DTT. This enzyme was activated in vitro more efficiently by reduced thioredoxin, which is a transducer of redox potential during illumination, than by DTT alone. Chloroplast thioredoxin-f activated the enzyme more efficiently than thioredoxin-m. The ACCase also was activated by thioredoxin reduced enzymatically with NADPH and NADP-thioredoxin reductase. These findings suggest that the reduction of ACCase is needed for activation of the enzyme, and a redox potential generated by photosynthesis is involved in its activation through thioredoxin as for enzymes of the reductive pentose phosphate cycle. The catalytic activity of ACCase was maximum at pH 8 and 2–5 mM Mg2+, indicating that light-produced changes in stromal pH and Mg2+ concentration modulate ACCase activity. These results suggest that light directly modulates a regulatory site of plastidic prokaryotic form of ACCase via a signal transduction pathway of a redox cascade and indirectly modulates its catalytic activity via stromal pH and Mg2+ concentration. A redox cascade is likely to link between light and fatty acid synthesis, resulting in coordination of fatty acid synthesis with photosynthesis.

cDNA cloning of Batis maritima methyl chloride transferase and purification of the enzyme

Methyl chloride transferase catalyzes the synthesis of methyl chloride from S-adenosine-l-methionine and chloride ion. This enzyme has been purified 2,700-fold to homogeneity from Batis maritima, a halophytic plant that grows abundantly in salt marshes. The purification of the enzyme was accomplished by a combination of ammonium sulfate fractionation, column chromatography on Sephadex G100 and adenosine-agarose, and TSK-250 size-exclusion HPLC. The purified enzyme exhibits a single band on SDS/PAGE with a molecular mass of approximately 22.5 kDa. The molecular mass of the purified enzyme was 22,474 Da as determined by matrix-associated laser desorption ionization mass spectrometry. The methylase can function in either a monomeric or oligomeric form. A 32-aa sequence of an internal fragment of the methylase was determined (GLVPGCGGGYDVVAMANPER FMVGLDIXENAL, where X represents unknown residue) by Edman degradation, and a full-length cDNA of the enzyme was obtained by rapid amplification of cDNA ends–PCR amplification of cDNA oligonucleotides. The cDNA gene contains an ORF of 690 bp encoding an enzyme of 230 aa residues having a predicted molecular mass of 25,761 Da. The disparity between the observed and calculated molecular mass suggests that the methylase undergoes posttranslational cleavage, possibly during purification. Sequence homologies suggest that the B. maritima methylase defines a new family of plant methyl transferases. A possible function for this novel methylase in halophytic plants is discussed.

Transmembrane topology of a CLC chloride channel

CLC chloride channels form a large and conserved gene family unrelated to other channel proteins. Knowledge of the transmembrane topology of these channels is important for understanding the effects of mutations found in human myotonia and inherited hypercalciuric kidney stone diseases and for the interpretation of structure–function studies. We now systematically study the topology of human ClC-1, a prototype CLC channel that is defective in human myotonia. Using a combination of in vitro glycosylation scanning and protease protection assays, we show that both N and C termini face the cytoplasm and demonstrate the presence of 10 (or less likely 12) transmembrane spans. Difficult regions were additionally tested by inserting cysteines and probing the effect of cysteine-modifying reagents on ClC-1 currents. The results show that D3 crosses the membrane and D4 does not, and that L549 between D11 and D12 is accessible from the outside. Further, since the modification of cysteines introduced between D11 and D12 and at the extracellular end of D3 strongly affect ClC-1 currents, these regions are suggested to be important for ion permeation.

Growth of Toxoplasma gondii is inhibited by aryloxyphenoxypropionate herbicides targeting acetyl-CoA carboxylase

Aryloxyphenoxypropionates, inhibitors of the plastid acetyl-CoA carboxylase (ACC) of grasses, also inhibit Toxoplasma gondii ACC. Clodinafop, the most effective of the herbicides tested, inhibits growth of T. gondii in human fibroblasts by 70% at 10 μM in 2 days and effectively eliminates the parasite in 2–4 days at 10–100 μM. Clodinafop is not toxic to the host cell even at much higher concentrations. Parasite growth inhibition by different herbicides is correlated with their ability to inhibit ACC enzyme activity, suggesting that ACC is a target for these agents. Fragments of genes encoding the biotin carboxylase domain of multidomain ACCs of T. gondii, Plasmodium falciparum, Plasmodium knowlesi, and Cryptosporidium parvum were sequenced. One T. gondii ACC (ACC1) amino acid sequence clusters with P. falciparum ACC, P. knowlesi ACC, and the putative Cyclotella cryptica chloroplast ACC. Another sequence (ACC2) clusters with that of C. parvum ACC, probably the cytosolic form.

Heteromultimeric CLC chloride channels with novel properties

The skeletal muscle chloride channel CLC-1 and the ubiquitous volume-activated chloride channel CLC-2 belong to a large gene family whose members often show overlapping expression patterns. CLC-1 and CLC-2 are coexpressed in skeletal and smooth muscle and in the heart. By coexpressing CLC-1 and CLC-2 in Xenopus oocytes, we now show the formation of novel CLC-1/CLC-2 heterooligomers that yield time-independent linear chloride currents with a chloride → bromide → iodide selectivity sequence. Formation of heterooligomeric CLC channels increases the number and possible functions of chloride channels.

Herbicide sensitivity determinant of wheat plastid acetyl-CoA carboxylase is located in a 400-amino acid fragment of the carboxyltransferase domain

A series of chimeral genes, consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) cDNA, and yeast ACC1 3′-tail, was used to complement a yeast ACC1 mutation. These genes encode a full-length plastid enzyme, with and without the putative chloroplast transit peptide, as well as five chimeric cytosolic/plastid proteins. Four of the genes, all containing at least half of the wheat cytosolic ACCase coding region at the 5′-end, complement the yeast mutation. Aryloxyphenoxypropionate and cyclohexanedione herbicides, at concentrations below 10 μM, inhibit the growth of haploid yeast strains that express two of the chimeric ACCases. This inhibition resembles the inhibition of wheat plastid ACCase observed in vitro and in vivo. The differential response to herbicides localizes the sensitivity determinant to the third quarter of the multidomain plastid ACCase. Sequence comparisons of different multidomain and multisubunit ACCases suggest that this region includes part of the carboxyltransferase domain, and therefore that the carboxyltransferase activity of ACCase (second half-reaction) is the target of the inhibitors. The highly sensitive yeast gene-replacement strains described here provide a convenient system to study herbicide interaction with the enzyme and a powerful screening system for new inhibitors.

Chloride is an allosteric effector of copper assembly for the yeast multicopper oxidase Fet3p: An unexpected role for intracellular chloride channels

GEF1 is a gene in Saccharomyces cerevisiae, which encodes a putative voltage-regulated chloride channel. gef1 mutants have a defect in the high-affinity iron transport system, which relies on the cell surface multicopper oxidase Fet3p. The defect is due to an inability to transfer Cu+ to apoFet3p within the secretory apparatus. We demonstrate that the insertion of Cu into apoFet3p is dependent on the presence of Cl−. Cu-loading of apoFet3p is favored at acidic pH, but in the absence of Cl− there is very little Cu-loading at any pH. Cl− has a positive allosteric effect on Cu-loading of apoFet3p. Kinetic studies suggest that Cl− may also bind to Fet3p and that Cu+ has an allosteric effect on the binding of Cl− to the enzyme. Thus, Cl− may be required for the metal loading of proteins within the secretory apparatus. These results may have implications in mammalian physiology, as mutations in human intracellular chloride channels result in disease.

Genetically determined chloride-sensitive hypertension and stroke

The stroke-prone spontaneously hypertensive rat (SHRSP) is a genetically determined model of “salt-sensitive” stroke and hypertension whose full phenotypic expression is said to require a diet high in Na+ and low in K+. We tested the hypothesis that dietary Cl− determines the phenotypic expression of the SHRSP. In the SHRSP fed a normal NaCl diet, supplementing dietary K+ with KCl exacerbated hypertension, whereas supplementing either KHCO3 or potassium citrate (KB/C) attenuated hypertension, when blood pressure (BP) was measured radiotelemetrically, directly and continually. Supplemental KCl, but not KB/C, induced strokes, which occurred in all and only those rats in the highest quartiles of both BP and plasma renin activity (PRA). PRA was higher with KCl than with KB/C. These observations demonstrate that with respect to both severity of hypertension and frequency of stroke the phenotypic expression of the SHRSP is (i) either increased or decreased, depending on whether the anionic component of the potassium salt supplemented is, or is not, Cl−; (ii) increased by supplementing Cl− without supplementing Na+, and despite supplementing K+; and hence (iii) both selectively Cl−-sensitive and Cl−-determined. The observations suggest that in the SHRSP selectively supplemented with Cl− the likelihood of stroke depends on the extent to which both BP and PRA increase.

Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase

To determine the mechanisms responsible for the termination of Ca2+-activated Cl− currents (ICl(Ca)), simultaneous measurements of whole cell currents and intracellular Ca2+ concentration ([Ca2+]i) were made in equine tracheal myocytes. In nondialyzed cells, or cells dialyzed with 1 mM ATP, ICl(Ca) decayed before the [Ca2+]i decline, whereas the calcium-activated potassium current decayed at the same rate as [Ca2+]i. Substitution of AMP-PNP or ADP for ATP markedly prolonged the decay of ICl(Ca), resulting in a rate of current decay similar to that of the fall in [Ca2+]i. In the presence of ATP, dialysis of the calmodulin antagonist W7, the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor KN93, or a CaMKII-specific peptide inhibitor the rate of ICl(Ca) decay was slowed and matched the [Ca2+]i decline, whereas H7, a nonspecific kinase inhibitor with low affinity for CaMKII, was without effect. When a sustained increase in [Ca2+]i was produced in ATP dialyzed cells, the current decayed completely, whereas in cells loaded with 5′-adenylylimidodiphosphate (AMP-PNP), KN93, or the CaMKII inhibitory peptide, ICl(Ca) did not decay. Slowly decaying currents were repeatedly evoked in ADP- or AMP-PNP-loaded cells, but dialysis of adenosine 5′-O-(3-thiotriphosphate) or okadaic acid resulted in a smaller initial ICl(Ca), and little or no current (despite a normal [Ca2+]i transient) with a second stimulation. These data indicate that CaMKII phosphorylation results in the inactivation of calcium-activated chloride channels, and that transition from the inactivated state to the closed state requires protein dephosphorylation.

The subcellular localization of acetyl-CoA carboxylase 2

Animals, including humans, express two isoforms of acetyl-CoA carboxylase (EC 6.4.1.2), ACC1 (Mr = 265 kDa) and ACC2 (Mr = 280 kDa). The predicted amino acid sequence of ACC2 contains an additional 136 aa relative to ACC1, 114 of which constitute the unique N-terminal sequence of ACC2. The hydropathic profiles of the two ACC isoforms generally are comparable, except for the unique N-terminal sequence in ACC2. The sequence of amino acid residues 1–20 of ACC2 is highly hydrophobic, suggesting that it is a leader sequence that targets ACC2 for insertion into membranes. The subcellular localization of ACC2 in mammalian cells was determined by performing immunofluorescence microscopic analysis using affinity-purified anti-ACC2-specific antibodies and transient expression of the green fluorescent protein fused to the C terminus of the N-terminal sequences of ACC1 and ACC2. These analyses demonstrated that ACC1 is a cytosolic protein and that ACC2 was associated with the mitochondria, a finding that was confirmed further by the immunocolocalization of a known human mitochondria-specific protein and the carnitine palmitoyltransferase 1. Based on analyses of the fusion proteins of ACC–green fluorescent protein, we concluded that the N-terminal sequences of ACC2 are responsible for mitochondrial targeting of ACC2. The association of ACC2 with the mitochondria is consistent with the hypothesis that ACC2 is involved in the regulation of mitochondrial fatty acid oxidation through the inhibition of carnitine palmitoyltransferase 1 by its product malonyl-CoA.

Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes

CD95/Fas/APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck. In vitro incubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30–65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.

Mutations in the chloride-bicarbonate exchanger gene AE1 cause autosomal dominant but not autosomal recessive distal renal tubular acidosis

Primary distal renal tubular acidosis (dRTA) is characterized by reduced ability to acidify urine, variable hyperchloremic hypokalemic metabolic acidosis, nephrocalcinosis, and nephrolithiasis. Kindreds showing either autosomal dominant or recessive transmission are described. Mutations in the chloride-bicarbonate exchanger AE1 have recently been reported in four autosomal dominant dRTA kindreds, three of these altering codon Arg589. We have screened 26 kindreds with primary dRTA for mutations in AE1. Inheritance was autosomal recessive in seventeen kindreds, autosomal dominant in one, and uncertain due to unknown parental phenotype or sporadic disease in eight kindreds. No mutations in AE1 were detected in any of the autosomal recessive kindreds, and analysis of linkage showed no evidence of linkage of recessive dRTA to AE1. In contrast, heterozygous mutations in AE1 were identified in the one known dominant dRTA kindred, in one sporadic case, and one kindred with two affected brothers. In the dominant kindred, the mutation Arg-589/Ser cosegregated with dRTA in the extended pedigree. An Arg-589/His mutation in the sporadic case proved to be a de novo mutation. In the third kindred, affected brothers both have an intragenic 13-bp duplication resulting in deletion of the last 11 amino acids of AE1. These mutations were not detected in 80 alleles from unrelated normal individuals. These findings underscore the key role of Arg-589 and the C terminus in normal AE1 function, and indicate that while mutations in AE1 cause autosomal dominant dRTA, defects in this gene are not responsible for recessive disease.