Expression and mutagenesis of ZntA, a zinc-transporting P-type ATPase from Escherichia coli.

Cation-transporting P-type ATPases comprise a major membrane protein family, the members of which are found in eukaryotes, eubacteria, and archaea. A phylogenetically old branch of the P-type ATPase family is involved in the transport of heavy-metal ions such as copper, silver, cadmium, and zinc. In humans, two homologous P-type ATPases transport copper. Mutations in the human proteins cause disorders of copper metabolism known as Wilson and Menkes diseases. E. coli possesses two genes for heavy-metal translocating P-type ATPases. We have constructed an expression system for one of them, ZntA, which encodes a 732 amino acid residue protein capable of transporting Zn(2+). A vanadate-sensitive, Zn(2+)-dependent ATPase activity is present in the membrane fraction of our expression strain. In addition to Zn(2+), the heavy-metal ions Cd(2+), Pb(2+), and Ag(+) activate the ATPase. Incubation of membranes from the expression strain with [gamma-(33)P]ATP in the presence of Zn(2+), Cd(2+), or Pb(2+) brings about phosphorylation of two membrane proteins with molecular masses of approximately 90 and 190 kDa, most likely representing the ZntA monomer and dimer, respectively. Although Cu(2+) can stimulate phosphorylation by [gamma-(33)P]ATP, it does not activate the ATPase. Cu(2+) also prevents the Zn(2+) activation of the ATPase when present in 2-fold excess over Zn(2+). Ag(+) and Cu(+) appear not to promote phosphorylation of the enzyme. To study the effects of Wilson disease mutations, we have constructed two site-directed mutants of ZntA, His475Gln and Glu470Ala, the human counterparts of which cause Wilson disease. Both mutants show a reduced metal ion stimulated ATPase activity (about 30-40% of the wild-type activity) and are phosphorylated much less efficiently by [gamma-(33)P]ATP than the wild type. In comparison to the wild type, the Glu470Ala mutant is phosphorylated more strongly by [(33)P]P(i), whereas the His475Gln mutant is phosphorylated more weakly. These results suggest that the mutation His475Gln affects the reaction with ATP and P(i) and stabilizes the enzyme in a dephosphorylated state. The Glu470Ala mutant seems to favor the E2 state. We conclude that His475 and Glu470 play important roles in the transport cycles of both the Wilson disease ATPase and ZntA.     

Metal-binding characteristics of the amino-terminal domain of ZntA: binding of lead is different compared to cadmium and zinc

ZntA from Escherichia coli, a P1-type ATPase, specifically transports Pb(II), Zn(II), and Cd(II). Most P1-type ATPases have an N-terminal domain that contains one or more copies of the conserved metal-binding motif, GXXCXXC. In ZntA, the N-terminal domain has approximately 120 residues with a single GXXCXXC motif, as well as four additional cysteine residues as part of the CCCDGAC motif. The metal-binding specificity and affinity of this domain in ZntA was investigated. Isolated proteins, N1-ZntA and N2-ZntA, containing residues 1-111 and 47-111 of ZntA, respectively, were characterized. N1-ZntA has both the CCCDGAC and GXXCXXC motifs, while N2-ZntA has only the GXXCXXC motif. ICP-MS measurements showed that N1-ZntA can bind both divalent metal ions such as Cd(II), Pb(II), and Zn(II) and monovalent metal ions such as Ag(I), with a stoichiometry of 1. N2-ZntA can bind Zn(II) and Cd(II) with a stoichiometry of 1 but not Pb(II). The affinity of N1-ZntA for Zn(II), Pb(II), and Cd(II) was measured by competition titration with metallochromic indicators. Association constants of approximately 10(8) M(-)(1) were obtained for Zn(II), Pb(II), and Cd(II) binding to N1-ZntA. To investigate whether the CCCDGAC sequence has an important role in binding specifically Pb(II), a mutant of ZntA, which lacked the first 46 residues, was constructed. This mutant, Delta46-ZntA, had the same activity as wtZntA with respect to Cd(II) and Zn(II). However, its activity with Pb(II) was similar to the mutant DeltaN-ZntA, which lacks the entire N-terminal domain (Mitra, B., and Sharma, R. (2001) Biochemistry 40, 7694-7699). Thus, binding of Pb(II) appears to involve different ligands, and possibly geometry, compared to Cd(II) and Zn(II).     

Structural basis for metal binding specificity: the N-terminal cadmium binding domain of the P1-type ATPase CadA

In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a betaalphabetabetaalphabeta fold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sphere was investigated by X-ray absorption spectroscopy. A novel metal binding environment was found, comprising the two conserved Cys residues of the metal binding motif and a Glu in loop 5. A bioinformatic search identifies an ensemble of highly homologous sequences presumably with the same function. Another group of highly homologous sequences is found which can be referred to as zinc-detoxifying P1-type ATPases with the metal binding pattern DCXXC in the N terminus. Because no carboxylate groups participate in Cu(I) or Ag(I) binding sites, we suggest that the acidic residue plays a key role in the coordination properties of divalent cations, hence conferring a function to the N terminus in the metal specificity of the ATPase.     

The membrane ATPase of alkalophilic Bacillus firmus RAB is an F1-type ATPase

At the optimal pH for growth (pH 10.5), alkalophilic Bacillus firmus RAB, an obligate aerobe, exhibits normal rates of oxidative phosphorylation despite the low transmembrane proton electrochemical gradient, about -60 mV (delta psi = -180 mV and delta pH = +120 mV). This bioenergetic problem might be resolved by use of an Na+ coupled ATP synthase; otherwise an F1F0-ATPase must be able to utilize low driving forces in this organism. The ATPase activity was extracted from everted membrane vesicles by low ionic strength treatment and purified to homogeneity by hydrophobic interaction chromatography and sucrose density gradient centrifugation. The ATPase preparation had the characteristic F1-ATPase subunit structure, with Mr values of 51,500 (alpha), 48,900 (beta), 34,400 (gamma), 23,300 (delta), and 14,500 (epsilon); the identity of the alpha and beta subunits was confirmed by immunoblotting with anti-beta of Escherichia coli and anti-B. firmus RAB F1. Methanol and octyl glucoside, agents that stimulated the low basal membrane ATPase activity 10- to 12-fold, dramatically elevated the MgATPase activity of the purified F1, more than 150-fold, to 50 mumol min-1 mg protein-1. Anti-F1 inhibited membrane ATPase activity greater than or equal to 80%. The membranes exhibited no Na+-stimulated or vanadate-sensitive ATPase activity when prepared in the absence or presence of Na+ or ATP. These findings, which are consistent with previous studies, establish that in alkalophilic bacteria, ATP hydrolysis, and presumably ATP synthesis is catalyzed by an F1F0-ATPase rather than a Na+ ATPase.     

The nucleosome-remodeling ATPase ISWI is regulated by poly-ADP-ribosylation

ATP-dependent nucleosome-remodeling enzymes and covalent modifiers of chromatin set the functional state of chromatin. However, how these enzymatic activities are coordinated in the nucleus is largely unknown. We found that the evolutionary conserved nucleosome-remodeling ATPase ISWI and the poly-ADP-ribose polymerase PARP genetically interact. We present evidence showing that ISWI is target of poly-ADP-ribosylation. Poly-ADP-ribosylation counteracts ISWI function in vitro and in vivo. Our work suggests that ISWI is a physiological target of PARP and that poly-ADP-ribosylation can be a new, important post-translational modification regulating the activity of ATP-dependent nucleosome remodelers.     

PICH, a centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for the spindle checkpoint

We identify PICH (Plk1-interacting checkpoint "helicase"), a member of the SNF2 ATPase family, as an interaction partner and substrate of Plk1. Following phosphorylation of PICH on the Cdk1 site T1063, Plk1 is recruited to PICH and controls its localization. Starting in prometaphase, PICH accumulates at kinetochores and inner centromeres. Moreover, it decorates threads that form during metaphase before increasing in length and progressively diminishing during anaphase. PICH-positive threads connect sister kinetochores and are dependent on tension, sensitive to DNase, and exacerbated in response to premature loss of cohesins or inhibition of topoisomerase II, suggesting that they represent stretched centromeric chromatin. Depletion of PICH causes the selective loss of Mad2 from kinetochores and completely abrogates the spindle checkpoint, resulting in massive chromosome missegregation. These data identify PICH as a novel essential component of checkpoint signaling. We propose that PICH binds to catenated centromere-related DNA to monitor tension developing between sister kinetochores.     

Cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation

Cytoplasmic dynein is a microtubule-associated motor that utilizes ATP hydrolysis to conduct minus-end directed transport of various organelles. Dynactin is a multisubunit complex that has been proposed to both link dynein with cargo and activate dynein motor function. The mechanisms by which dynactin regulates dynein activity are not clear. In this study, we examine the role of dynactin in regulating dynein ATPase activity. We show that dynein-microtubule binding and ATP-dependent release of dynein from microtubules are reduced in dynactin null mutants, Deltaro-3 (p150(Glued)) and Deltaro-4 (Arp1), relative to wild-type. The dynein-microtubule binding activity, but not the ATP-dependent release of dynein from microtubules, is restored by in vitro mixing of extracts from dynein and dynactin mutants. Dynein produced in a Deltaro-3 mutant has approximately 8-fold reduced ATPase activity relative to dynein isolated from wild-type. However, dynein ATPase activity from wild-type is not reduced when dynactin is separated from dynein, suggesting that dynein produced in a dynactin mutant is inactivated. Treatment of dynein isolated from the Deltaro-3 mutant with lambda protein phosphatase restores the ATPase activity to near wild-type levels. The reduced dynein ATPase activity observed in dynactin null mutants is mainly due to altered affinity for ATP. Radiolabeling experiments revealed that low molecular mass proteins, particularly 20- and 8-kDa proteins, that immunoprecipitate with dynein heavy chain are hyperphosphorylated in the dynactin mutant and dephosphorylated upon lambda protein phosphatase treatment. The results suggest that cytoplasmic dynein ATPase activity is regulated by dynactin-dependent phosphorylation of dynein light chains.     

Tumor necrosis factor receptor 1 is an ATPase regulated by silencer of death domain

Self-aggregation of tumor necrosis factor receptor type 1 (TNFR1) induces spontaneous downstream signaling and results in cell death. It has been suggested that silencer of death domain (SODD) binds TNFR1 monomers to prevent self-aggregation. We found that SODD binds through its BAG domain to the ATPase domain of Hsp70. We also determined that SODD binds through its BAG domain to TNFR1. ATP, but not nonhydrolyzable ATP-gamma S, regulates the SODD binding by Hsp70 or TNFR1. ATP binding by TNFR1 was abolished when a point mutation was introduced into a phosphate-binding loop motif characteristic of ATP-binding proteins, suggesting that TNFR1 functions as an ATPase. Furthermore, TNFR1 was present in aggregates in ATP-depleted cells and SODD disassembled aggregates in vitro only in the presence of ATP. These data suggest that SODD functions as a cofactor analogous to the nucleotide exchange factor BAG-1, which modulates the ATPase cycle of Hsp70 proteins. We propose a new model in which a nucleotide-dependent conformational change in TNFR1 has a key role in regulating TNF signaling.     

The metal-binding sites of the zinc-transporting P-type ATPase of Escherichia coli. Lys and Asp in the seventh and eighth transmembrane segments of ZntA contribute to the coupling of metal binding and ATPase activity

ZntA is a P-type ATPase which transports Zn²⁺, Pb²⁺ and Cd²⁺ out of the cell. Two cysteine-containing motifs, CAAC near the N-terminus and CPC in transmembrane helix 6, are involved in binding of the translocated metal. We have studied these motifs by mutating the cysteines to serines. The roles of two other possible metal-binding residues, K⁶⁹³ and D⁷¹⁴, in transmembrane helices 7 and 8, were also addressed. The mutation CAAC → SAAS reduces the ATPase activity by 50%. The SAAS mutant is phosphorylated with ATP almost as efficiently as the wild type. However, its phosphorylation with P_i is poorer than that of the wild type and its dephosphorylation rate is faster than that of the wild type ATPase. The CPC → SPS mutant is inactive but residual phosphorylation with ATP could still be observed. The most important findings of this work deal with the prospective metal-binding residues K⁶⁹³ and D⁷¹⁴: the substitution K693N eliminates the Zn²⁺-stimulated ATPase activity completely, although significant Zn²⁺-dependent phosphorylation by ATP remains. The K693N ATPase is hyperphosphorylated by P_i. ZntA carrying the change D714M has strong metal-independent ATPase activity and is very weakly phosphorylated both by ATP and P_i. In conclusion, K⁶⁹³ and D⁷¹⁴ are functionally essential and appear to contribute to the metal specificity of ZntA, most probably by being parts of the metal-binding site made up by the CPC motif.     

Streptococcal cytoplasmic pH is regulated by changes in amount and activity of a proton-translocating ATPase

The Streptococcus faecalis H+-ATPase (F1 X F0 complex) level was elevated when the cytoplasmic pH was shifted below 7.5. The elevated level was attained by the increase in functional unit (F1 X F0 complex) in membranes, but not by the activation of the enzyme. Our data strongly suggested that the increase in enzyme arises from stimulation of enzyme biosynthesis. When calls growing at pH 7.6 were transferred to an acid medium with a pH below 7, the amount of H+-ATPase increased. The amount of H+-ATPase decreased to the basal level when the medium was alkalized again. Cytoplasmic pH was not controlled normally in cells where a change in the amount of H+-ATPase was inhibited. Based on these findings and previous data (Kobayashi, H. (1985) J. Biol. Chem. 260, 72-76), we propose a model for the regulatory mechanism of streptococcal cytoplasmic pH: the pH is regulated by changes in amount and activity of the H+-ATPase, which are dependent on the cytoplasmic pH.     

The metal-binding sites of the zinc-transporting P-type ATPase of Escherichia coli. Lys693 and Asp714 in the seventh and eighth transmembrane segments of ZntA contribute to the coupling of metal binding and ATPase activity

ZntA is a P-type ATPase which transports Zn(2+), Pb(2+) and Cd(2+) out of the cell. Two cysteine-containing motifs, CAAC near the N-terminus and CPC in transmembrane helix 6, are involved in binding of the translocated metal. We have studied these motifs by mutating the cysteines to serines. The roles of two other possible metal-binding residues, K(693) and D(714), in transmembrane helices 7 and 8, were also addressed. The mutation CAAC-->SAAS reduces the ATPase activity by 50%. The SAAS mutant is phosphorylated with ATP almost as efficiently as the wild type. However, its phosphorylation with P(i) is poorer than that of the wild type and its dephosphorylation rate is faster than that of the wild type ATPase. The CPC-->SPS mutant is inactive but residual phosphorylation with ATP could still be observed. The most important findings of this work deal with the prospective metal-binding residues K(693) and D(714): the substitution K693N eliminates the Zn(2+)-stimulated ATPase activity completely, although significant Zn(2+)-dependent phosphorylation by ATP remains. The K693N ATPase is hyperphosphorylated by P(i). ZntA carrying the change D714M has strong metal-independent ATPase activity and is very weakly phosphorylated both by ATP and P(i). In conclusion, K(693) and D(714) are functionally essential and appear to contribute to the metal specificity of ZntA, most probably by being parts of the metal-binding site made up by the CPC motif.     

Expression of P2X6, a purinergic receptor subunit,is affected by dietary zinc deficiency in rat hippocampus

The purpose of these experiments was to determine whether dietary zinc depletion affected protein expression in the hippocampus. Eleven weanling Sprague-Dawley male rats (21 d) were fed the AIN-93G diet containing 1.5 ppm zinc and supplemented with 30 ppm of zinc in the drinking water. After 1 wk, the rats were randomly divided into three groups: control (n=3), pair fed (n=3), and zinc restricted (n=5). All groups consumed the same diet. The zinc-restricted group consumed water containing no zinc. The rats were sacrificed 3 wk later. Chelatable zinc levels in the hippocampus, as measured by N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ) staining, were significantly reduced in the zinc-restricted group. Analysis of hippocampal protein expression by two-dimensional electrophoresis (2DE) revealed increased expression of the P2X₆ purinergic receptor in the zinc-restricted rats, as determined by MALDI mass spectrometry (MS) and database analysis. The data provided evidence for the dual effects of dietary zinc deficiency on the hippocampus, reducing ionic zinc levels and stimulating protein expression. The role the P2X₆ receptor plays in the physiological response of the hippocampus to zinc depletion remains to be determined.     

Fragile X protein family member FXR1P is regulated by microRNAs

FXR1P is one of two autosomal paralogs of the fragile X mental retardation protein FMRP. The absence of FMRP causes fragile X syndrome, the leading cause of hereditary mental retardation. FXR1P plays an important role in normal muscle development and has been implicated in facioscapulohumeral muscular dystrophy (FSHD). Its absence also causes cardiac abnormalities in both mice and zebrafish. To examine miRNA-mediated regulation of FMRP and FXR1P, we studied their expression in a conditional Dicer knockdown cell line, DT40. We found that FXR1P, but not FMRP, is significantly increased upon Dicer knockdown and the consequent reduction of miRNAs, suggesting that FXR1P is regulated by miRNAs while FMRP is not in DT40 cells. Expression of a luciferase reporter bearing the 3′ untranslated region (3′UTR) of FXR1 was significantly increased in the absence of miRNAs, confirming miRNA-mediated regulation of FXR1P, while a luciferase reporter bearing the FMR1 3′UTR was not. We identified one of the regulatory regions in the 3′UTR of FXR1 by removing a conserved, 8-nucleotide miRNA seed sequence common to miRNAs 25, 32, 92, 363, and 367 and demonstrated loss of miRNA-mediated suppression. Treatment with specific miRNA hairpin inhibitors to each of the miRNAs in the seed sequence showed that miRs 92b, 363, and 367 regulated FXR1P expression. Accordingly, overexpression of the miRNA 367 mimic significantly decreased endogenous FXR1P expression in human cell lines HEK-293T and HeLa. We report for the first time that FXR1P is regulated through miRNA binding, with one site being the miR-25/32/92/363/367 seed sequence.     

Expression of a vegetative-storage-protein gene from Arabidopsis is regulated by copper, senescence and ozone

Emerging data suggest that the mechanisms regulating plant copper homeostasis could be implicated in stress and senescence signal transduction pathways. To gain insight into copper-modulated patterns of gene expression, copper-treated Arabidopsis thaliana (L.) Heynh. plants were analysed by mRNA differential display. The experimental conditions were selected using aggregation of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) as a molecular sensor to monitor copper-induced oxidative stress. Two copper-induced messengers encoding a vegetative storage protein (VSP2) were isolated by this technique. Both clones differed in the length of their 3'-untranslated region according to the presence of two polyadenylation signals in this region. VSP2 expression was further studied under natural senescence and various conditions causing oxidative stress, such as ozone exposure, paraquat and H2O2 treatments. The expression of other messengers related to copper homeostasis and detoxification processes was followed in parallel to that of VSP2. Here, we describe specific gene-expression responses to copper treatment, and present arguments connecting copper homeostasis, senescence and antioxidative responses in plants. Our results are consistent with the role of VSPs as temporary nitrogen-storage proteins which accumulate if nutrients are abundant, either in developing organs or in cotyledons and mature leaves subjected to generalized protein mobilization, such as those conditions created under severe oxidative stress.