Cloning of a bovine orphan transporter and its short splicing variant.
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We have isolated a cDNA (bv7-3) encoding a member of the Na+,Cl(-)-dependent transporter family and its short splicing variant (bv7-3s) by screening a bovine retina cDNA library. Sequence analysis revealed that bv7-3 encodes a protein of 729 amino acids and is a bovine homologue of the rat orphan transporter v7-3-2. bv7-3s contains 265 amino acids, sharing 252 N-terminal amino acids with bv7-3. Both mRNAs for bv7-3 and bv7-3s were detected in nervous system by Northern blot analysis. In immunofluorescence analysis in transfected HEK 293T cells, myc-tagged bv7-3 was mainly detected on the plasma membrane, whereas myc-tagged bv7-3s showed a pattern of intracellular membrane staining. (+info)
Asp45 is a Mg2+ ligand in the ArsA ATPase.
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The ATPase activity of ArsA, the catalytic subunit of the plasmid-encoded, ATP-dependent extrusion pump for arsenicals and antimonials in Escherichia coli, is allosterically activated by arsenite or antimonite. Magnesium is essential for ATPase activity. To examine the role of Asp45, mutants were constructed in which Asp45 was changed to Glu, Asn, or Ala. Cells expressing these mutated arsA genes lost arsenite resistance to varying degrees. Purified D45A and D45N enzymes were inactive. The purified D45E enzyme exhibited approximately 5% of the wild type activity with about a 5-fold decrease in affinity for Mg2+. Intrinsic tryptophan fluorescence was used to probe Mg2+ binding. ArsA containing only Trp159 exhibited fluorescence enhancement upon the addition of MgATP, which was absent in D45N and D45A. As another measure of conformation, limited trypsin digestion was used to estimate the surface accessibility of residues in ArsA. ATP and Sb(III) synergistically protected wild type ArsA from trypsin digestion. Subsequent addition of Mg2+ increased trypsin sensitivity. D45N and D45A remained protected by ATP and Sb(III) but lost the Mg2+ effect. D45E exhibited an intermediate Mg2+ response. These results indicate that Asp45 is a Mg2+-responsive residue, consistent with its function as a Mg2+ ligand. (+info)
The ATPase mechanism of ArsA, the catalytic subunit of the arsenite pump.
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The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein. The single tryptophan-containing Trp159 ArsA was used to elucidate the elementary steps of the ATPase mechanism by fluorescence stopped-flow experiments. The binding and hydrolysis of MgATP is a multistep process with a minimal kinetic mechanism (Mechanism 1). A notable feature of the reaction is that MgATP binding induces a slow transient increase in fluorescence of ArsA, which is independent of the ATP concentration, indicative of the build-up of a pre-steady state intermediate. This finding, coupled with a phosphate burst, implies that the steady-state intermediate builds up subsequent to product release. We propose that the rate-limiting step is an isomerization between different conformational forms of ArsA. kcat is faster than the phosphate burst, indicating that both nucleotide binding sites of ArsA are catalytic. Consistent with this interpretation, approximately 2 mol of phosphate are released per mole of ArsA during the phosphate burst. (+info)
Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila.
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Macrophages in the Drosophila embryo are responsible for the phagocytosis of apoptotic cells and are competent to engulf bacteria. Croquemort (CRQ) is a CD36-related receptor expressed exclusively on these macrophages. Genetic evidence showed that crq was essential for efficient phagocytosis of apoptotic corpses but was not required for the engulfment of bacteria. The expression of CRQ was regulated by the amount of apoptosis. These data define distinct pathways for the phagocytosis of corpses and bacteria in Drosophila. (+info)
Functional complementation of the malvolio mutation in the taste pathway of Drosophila melanogaster by the human natural resistance-associated macrophage protein 1 (Nramp-1).
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The malvolio (mvl) gene of Drosophila melanogaster encodes a protein with a high degree of homology to natural resistance-associated macrophage proteins (Nramps). This family of integral membrane proteins, many of which appear to function as cation transporters, is remarkably conserved in several phylogenetically distinct species. In Drosophila melanogaster, the protein Mvl is expressed in macrophages and in differentiated neurons; loss-of-function mutations lead to defects in gustatory behaviour. The human Nramp-1 protein was expressed in Drosophila melanogaster using the hsp70 promoter. Overexpression in normal animals does not lead to any alterations in their behaviour or physiology. In mutants, however, ubiquitous expression of human Nramp-1 can totally rescue the taste defect. This finding that Nramp-1 can complement the taste defect in mvl mutants provides a potent means of exploiting behavioural genetics to dissect the function of Nramp-1 and to identify other molecules involved with this transport system. (+info)
The anion-stimulated ATPase ArsA shows unisite and multisite catalytic activity.
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ArsA, an anion-stimulated ATPase, consists of two nucleotide binding domains, A1 in the N terminus and A2 in the C terminus of the protein, connected by a linker. The A1 domain contains a high affinity ATP binding site, whereas the A2 domain has low affinity and it requires the allosteric ligand antimonite for binding ATP. ArsA is known to form a UV-activated adduct with [alpha-(32)P]ATP in the linker region. This study shows that on addition of antimonite, much more adduct is formed. Characterization of the nature of the adduct suggests that it is between ArsA and ADP, instead of ATP, indicating that the adduct formation reflects hydrolysis of ATP. The present study also demonstrates that the A1 domain is capable of carrying out unisite catalysis in the absence of antimonite. On addition of antimonite, multisite catalysis involving both A1 and A2 sites occurs, resulting in a 40-fold increase in ATPase activity. Studies with mutant proteins suggest that the A2 site may be second in the sequence of events, so that its role in catalysis is dependent on a functional A1 site. It is also proposed that ArsA goes through an ATP-bound and an ADP-bound conformation, and the linker region, where ADP binds under both unisite and multisite catalytic conditions, may play an important role in the energy transduction process. (+info)
Studies on the ADP-ribose pyrophosphatase subfamily of the nudix hydrolases and tentative identification of trgB, a gene associated with tellurite resistance.
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Four Nudix hydrolase genes, ysa1 from Saccharomyces cerevisiae, orf209 from Escherichia coli, yqkg from Bacillus subtilis, and hi0398 from Hemophilus influenzae were amplified, cloned into an expression vector, and transformed into E. coli. The expressed proteins were purified and shown to belong to a subfamily of Nudix hydrolases active on ADP-ribose. Comparison with other members of the subfamily revealed a conserved proline 16 amino acid residues downstream of the Nudix box, common to all of the ADP-ribose pyrophosphatase subfamily. In this same region, a conserved tyrosine designates another subfamily, the diadenosine polyphosphate pyrophosphatases, while an array of eight conserved amino acids is indicative of the NADH pyrophosphatases. On the basis of these classifications, the trgB gene, a tellurite resistance factor from Rhodobacter sphaeroides, was predicted to designate an ADP-ribose pyrophosphatase. In support of this hypothesis, a highly specific ADP-ribose pyrophosphatase gene from the archaebacterium, Methanococcus jannaschii, introduced into E. coli, increased the transformant's tolerance to potassium tellurite. (+info)
Mechanism of the ArsA ATPase.
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The ArsAB ATPase confers metalloid resistance in Escherichia coli by pumping toxic anions out of the cells. This transport ATPase shares structural and perhaps mechanism features with ABC transporters. The ArsAB pump is composed of a membrane subunit that has two groups of six transmembrane segments, and the catalytic subunit, the ArsA ATPase. As is the case with many ABC transporters, ArsA has an internal repeat, each with an ATP binding domain, and is allosterically activated by substrates of the pump. The mechanism of allosteric activation of the ArsA ATPase has been elucidated at the molecular level. Binding of the activator produces a conformational change that forms a tight interface of the nucleotide binding domains. In the rate-limiting step in the overall reaction, the enzyme undergoes a slow conformational change. The allosteric activator accelerates catalysis by increasing the velocity of this rate-limiting step. We postulate that similar conformational changes may be rate-limiting in the mechanism of ABC transporters. (+info)