RESUMEN
The Zap1 transcriptional activator from Saccharomyces cerevisiae induces expression of a series of genes containing an 11 base pair conserved promoter element (ZRE) under conditions of zinc deficiency. This work shows that Zap1 uses four of its seven zinc finger domains to contact the ZRE and that two of these dominate the interaction by contacting the essential ACC-GGT ends. Two Zn finger domains (ZF1 and ZF2) do not contact DNA, and a third ZF3 may be more important for interfinger protein-protein interactions. Zn finger domains important for ZRE contact were identified from triple mutations in Zap1, changing three residues in the alpha helix in each finger known to be important for DNA contacts in Zn finger proteins. Replacement of -1, 3, and 6 helix residues in ZF4 and ZF7 reduced the affinity of Zap1 for the wild-type ZRE. In contrast, triple mutations within the intervening ZF5 and ZF6 domains had minimal effect. The data argue that fingers 4 and 7 contact the ACC-GGT ends while fingers 5 and 6 contact the 5 bp central ZRE sequence. This conclusion is corroborated by decreased Zap1 affinity for a ZRE DNA duplex containing mutations of the AC-GT ends of the ZRE, whereas transversion mutations within the central 5 bp of the ZRE had minimal effect on Zap1 binding affinity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/metabolismo , Dedos de Zinc , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Conejos , Elementos de Respuesta/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Resonancia por Plasmón de Superficie , Transactivadores/química , Transactivadores/genética , Factores de Transcripción , Dedos de Zinc/genéticaRESUMEN
Iron homeostasis in the yeast Saccharomyces cerevisiae is regulated at the transcriptional level by Aft1p, which activates the expression of its target genes in response to low-iron conditions. The yeast genome contains a paralog of AFT1, which has been designated AFT2. To establish whether AFT1 and AFT2 have overlapping functions, a mutant containing a double aft1Deltaaft2Delta deletion was generated. Growth assays established that the single aft2Delta strain exhibited no iron-dependent phenotype. However, the double-mutant aft1Deltaaft2Delta strain was more sensitive to low-iron growth conditions than the single-mutant aft1Delta strain. A mutant allele of AFT2 (AFT2-1(up)), or overexpression of the wild-type AFT2 gene, led to partial complementation of the respiratory-deficient phenotype of the aft1Delta strain. The AFT2-1(up) allele also increased the uptake of (59)Fe in an aft1Delta strain. DNA microarrays were used to identify genes regulated by AFT2. Some of the AFT2-regulated genes are known to be regulated by Aft1p; however, AFT2-1(up)-dependent activation was independent of Aft1p. The kinetics of induction of two genes activated by the AFT2-1(up) allele are consistent with Aft2p acting as a direct transcriptional factor. Truncated forms of Aft1p and Aft2p bound to a DNA duplex containing the Aft1p binding site in vitro. The wild-type allele of AFT2 activated transcription in response to growth under low-iron conditions. Together, these data suggest that yeast has a second regulatory pathway for the iron regulon, with AFT1 and AFT2 playing partially redundant roles.
Asunto(s)
Proteínas Fúngicas/metabolismo , Hierro/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , ADN de Hongos/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Expresión Génica , Genes Fúngicos , Homeostasis , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Regiones Promotoras Genéticas , Unión Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genéticaRESUMEN
Sco1 is a conserved essential protein, which has been implicated in the delivery of copper to cytochrome c oxidase, the last enzyme of the electron transport chain. In this study, we show for the first time that the purified C-terminal domain of yeast Sco1 binds one Cu(I)/monomer. X-ray absorption spectroscopy suggests that the Cu(I) is ligated via three ligands, and we show that two cysteines, present in a conserved motif CXXXC, and a conserved histidine are involved in Cu(I) ligation. The mutation of any one of the conserved residues in Sco1 expressed in yeast abrogates the function of Sco1 resulting in a non-functional cytochrome c oxidase complex. Thus, the function of Sco1 correlates with Cu(I) binding. Data obtained from size-exclusion chromatography experiments with mitochondrial lysates suggest that full-length Sco1 may be oligomeric in vivo.
Asunto(s)
Proteínas Portadoras/química , Cobre/metabolismo , Complejo IV de Transporte de Electrones/fisiología , Proteínas de la Membrana/química , Secuencias de Aminoácidos , Dimerización , Ligandos , Proteínas de la Membrana/fisiologíaRESUMEN
The Saccharomyces cerevisiae genome contains a predicted gene, YPR008w, homologous to the gene encoding the copper-activated transcription factor Ace1. The product of the YPR008w gene, designated Haa1, regulates the transcription of a set of yeast genes, many of which encode membrane proteins. Two main target genes of Haa1 are the multidrug resistance gene YGR138c and the YRO2 homolog to the plasma membrane Hsp30. Haa1 is localized to the nucleus. Haa1-induced expression of YGR138c and YRO2 appears to be direct. Induction of HAA1 using a GAL1/HAA1 fusion gene resulted in rapid galactose-induced expression of both HAA1 and target genes. Although Haa1 has a sequence very similar to the Cu-activated DNA binding domain of Ace1, expression of Haa1 target genes was found to be independent of the copper status of cells. Haa1 does not exhibit metalloregulation in cells incubated with a range of transition metal salts. Haa1 does not exhibit any cross-talk with Ace1. Overexpression of Haa1 does not compensate for cells lacking a functional Ace1. The lack of metalloregulation of Haa1 despite the strong sequence similarity to the copper regulatory domain of Ace1 is discussed.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Transactivadores/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Cobre/metabolismo , Cisteína/química , Proteínas de Unión al ADN/metabolismo , Diploidia , Galactosa/metabolismo , Vectores Genéticos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Zinc/metabolismoRESUMEN
Cox17 is the candidate copper metallochaperone for delivery of copper ions to the mitochondrion for assembly of cytochrome c oxidase. Cox17 purified as a recombinant molecule lacking any purification tag binds three Cu(I) ions per monomer in a polycopper cluster as shown by X-ray absorption spectroscopy. The CuCox17 complex exists in a dimer/tetramer equilibrium with a 20 microM k(d). The spectroscopic data do not discern whether the dimeric complex forms a single hexanuclear Cu(I) cluster or two separate trinuclear Cu(I) clusters. The Cu(I) cluster(s) exhibit(s) predominantly trigonal Cu(I) coordination. The cluster(s) in Cox17 resemble(s) the polycopper clusters in Ace1 and the Cup1 metallothionein in being pH-stable and luminescent. The physical properties of the CuCox17 complex purified as an untagged molecule differ from those reported previously for a GST-Cox17 fusion protein. The CuCox17 cluster is distinct from the polycopper cluster in Cup1 in being labile to ligand exchange. CuCox17 localized within the intermitochondrial membrane space appears to be predominantly tetrameric, whereas the cytosolic CuCox17 is primarily a dimeric species. Cys-->Ser substitutions at Cys23, Cys24, or Cys26 abolish the Cox17 function and prevent tetramerization, although Cu(I) binding is largely unaffected. Thus, the oligomeric state of Cox17 may be important to its physiological function.
Asunto(s)
Proteínas de Transporte de Catión , Cobre/química , Metaloproteínas/química , Mitocondrias/química , Proteínas/química , Western Blotting , Cisteína/genética , Dimerización , Ditiotreitol/química , Complejo IV de Transporte de Electrones/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Glutatión Transferasa/genética , Sustancias Macromoleculares , Metaloproteínas/biosíntesis , Metaloproteínas/genética , Metaloproteínas/aislamiento & purificación , Metalotioneína/química , Mitocondrias/enzimología , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/aislamiento & purificación , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Biosíntesis de Proteínas , Proteínas/genética , Proteínas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Espectrofotometría Ultravioleta , Análisis Espectral , Rayos XRESUMEN
Human neuronal growth inhibitory factor, a metalloprotein classified as metallothionein-3 (MT-3), impairs the survival and the neurite formation of cultured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and single mutants P7S and P9A of human Zn(7)-MT-3 were generated, and their effects on the biological activity and the structure of the protein were examined. The biological results clearly established the necessity of both proline residues for the inhibitory activity, as even single mutants were found to be inactive. Using electronic absorption, circular dichroism (CD), magnetic CD (MCD), and (113)Cd NMR spectroscopy, the structural features of the metal-thiolate clusters in the double mutant Cd(7)-mutMT-3 were investigated and compared with those of wild-type Cd(7)-MT-3 [Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vasák, M. (1999) Biochemistry 38, 10158] and the well characterized Cd(7)-MT-2a from rabbit liver. Similarly to (113)Cd(7)-MT-3 the (113)Cd NMR spectrum of (113)Cd(7)-mutMT-3 at 298 K revealed four major and three minor resonances (approximately 20% of the major ones) between 590 and 680 ppm, originating from a Cd(4)S(11) cluster in the alpha-domain and a Cd(3)S(9) cluster in the beta-domain, respectively. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all resonances showed the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in (113)Cd(7)-mutMT-3 the temperature rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd(3)S(9) cluster resonances, no such temperature effect was observed in (113)Cd(7)-MT-3. To account for the observed NMR features, a dynamic structural model for the beta-domain is proposed, which involves a folded and a partially unfolded state. It is suggested that in the partially unfolded state a slow cis/trans isomerization of Cys-Pro(7) or Cys-Pro(9) amide bonds in (113)Cd(7)-MT-3 takes place and that this process represents a rate-limiting step in a correct domain refolding. In addition, closely similar apparent stability constants of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. These results suggest that specific structural features dictated by the repetitive (Cys-Pro)(2) sequence in the beta-domain of MT-3 and not its altered metal binding affinity compared to MT-1/MT-2 isoforms are responsible for the biological activity of this protein.
Asunto(s)
Secuencia Conservada , Ácido Egtácico/análogos & derivados , Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/genética , Metalotioneína/química , Metalotioneína/genética , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Prolina/química , Secuencia de Aminoácidos , Animales , Cadmio/metabolismo , Supervivencia Celular/fisiología , Células Cultivadas , Quelantes/química , Dicroismo Circular , Ácido Egtácico/química , Radioisótopos de Flúor , Inhibidores de Crecimiento/metabolismo , Inhibidores de Crecimiento/fisiología , Humanos , Concentración de Iones de Hidrógeno , Isótopos , Metalotioneína/metabolismo , Metalotioneína/fisiología , Metalotioneína 3 , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Resonancia Magnética Nuclear Biomolecular , Prolina/genética , Unión Proteica/genética , Conejos , Ratas , Espectrofotometría , Relación Estructura-Actividad , Volumetría , Zinc/metabolismoRESUMEN
The copper metallochaperone Cox17 is proposed to shuttle Cu(I) ions to the mitochondrion for the assembly of cytochrome c oxidase. The Cu(I) ions are liganded by cysteinyl thiolates. Mutational analysis on the yeast Cox17 reveals three of the seven cysteinyl residues to be critical for Cox17 function, and these three residues are present in a Cys-Cys-Xaa-Cys sequence motif. Single substitution of any of these three cysteines with serines results in a nonfunctional cytochrome oxidase complex. Cells harboring such a mutation fail to grow on nonfermentable carbon sources and have no cytochrome c oxidase activity in isolated mitochondria. Wild-type Cox17 purified as untagged protein binds three Cu(I) ions/molecule. Mutant proteins lacking only one of these critical Cys residues retain the ability to bind three Cu(I) ions and are imported within the mitochondria. In contrast, Cox17 molecules with a double Cys --> Ser mutation exhibit no Cu(I) binding but are still localized to the mitochondria. Thus, mitochondrial uptake of Cox17 is not restricted to the Cu(I) conformer of Cox17. COX17 was originally cloned by virtue of complementation of a mutant containing a nonfunctional Cys --> Tyr substitution at codon 57. The mutant C57Y Cox17 fails to accumulate within the mitochondria but retains the ability to bind three Cu(I) ions. A C57S Cox17 variant is functional, and a quadruple Cox17 mutant with C16S/C36S/C47S/C57S substitutions binds three Cu(I) ions. Thus, only three cysteinyl residues are important for the ligation of three Cu(I) ions. A novel mode of Cu(I) binding is predicted.
Asunto(s)
Proteínas de Transporte de Catión , Análisis Mutacional de ADN , Mitocondrias/metabolismo , Proteínas/genética , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Proteínas Transportadoras de Cobre , Chaperonas Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas/química , Saccharomyces cerevisiae/genéticaRESUMEN
In Saccharomyces cerevisiae, copper ions regulate gene expression through the two transcriptional activators, Ace1 and Mac1. Ace1 mediates copper-induced gene expression in cells exposed to stressful levels of copper salts, whereas Mac1 activates a subset of genes under copper-deficient conditions. DNA microarray hybridization experiments revealed a limited set of yeast genes differentially expressed under growth conditions of excess copper or copper deficiency. Mac1 activates the expression of six S. cerevisiae genes, including CTR1, CTR3, FRE1, FRE7, YFR055w, and YJL217w. Two of the last three newly identified Mac1 target genes have no known function; the third, YFR055w, is homologous to cystathionine gamma-lyase encoded by CYS3. Several genes that are differentially expressed in cells containing a constitutively active Mac1, designated Mac1(up1), are not direct targets of Mac1. Induction or repression of these genes is likely a secondary effect of cells because of constitutive Mac1 activity. Elevated copper levels induced the expression of the metallothioneins CUP1 and CRS5 and two genes, FET3 and FTR1, in the iron uptake system. Copper-induced FET3 and FTR1 expression arises from an indirect copper effect on cellular iron pools.
Asunto(s)
Cobre/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genes Fúngicos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas Portadoras , Cobre/deficiencia , Cobre/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Hierro/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , Proteínas Nucleares/metabolismo , Ensayos de Protección de Nucleasas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Endonucleasas Específicas del ADN y ARN con un Solo Filamento/metabolismo , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The Zap1 transcriptional activator of Saccharomyces cerevisiae controls zinc homeostasis. Zap1 induces target gene expression in zinc-limited cells and is repressed by high zinc. One such target gene is ZAP1 itself. In this report, we examine how zinc regulates Zap1 function. First, we show that transcriptional autoregulation of Zap1 is a minor component of zinc responsiveness; most regulation of Zap1 activity occurs post-translationally. Secondly, nuclear localization of Zap1 does not change in response to zinc, suggesting that zinc regulates DNA binding and/or activation domain function. To understand how Zap1 responds to zinc, we performed a functional dissection of the protein. Zap1 contains two activation domains. DNA-binding activity is conferred by five C-terminal C(2)H(2) zinc fingers and each finger is required for high-affinity DNA binding. The zinc-responsive domain of Zap1 also maps to the C-terminal zinc fingers. Furthermore, mutations that disrupt some of these fingers cause constitutive activity of a bifunctional Gal4 DNA-binding domain-Zap1 fusion protein. These results demonstrate a novel function of Zap1 zinc fingers in zinc sensing as well as DNA binding.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Dedos de Zinc/fisiología , Zinc/metabolismo , Núcleo Celular/química , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Técnica del Anticuerpo Fluorescente , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genes Reporteros/genética , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Biosíntesis de Proteínas/efectos de los fármacos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Transducción de Señal/efectos de los fármacos , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Zinc/deficiencia , Zinc/farmacología , Dedos de Zinc/genéticaRESUMEN
Mac1 is a transcriptional activator whose activity is inhibited by copper ions. Mutagenesis studies were carried out to map residues important in the copper inhibition of Mac1 activity. Seven new missense mutations were identified that resulted in copper-independent Mac1 transcriptional activation. All seven mutations were clustered in one of two C-terminal cysteine-rich motifs, designated the C1 motif. All but one of the constitutive Mac1 mutations occurred in one of the conserved six residues in the (264)CXC[(X)(4)]CXC[(X)(2)]C[(X)(2)][H(279)]C1 motif. The lone exception was a L260S substitution. Two additional MAC1 mutations exhibiting constitutive activity were in-frame deletions encompassing portions C1. Engineered mutations in the second cysteine-rich motif did not yield a constitutively active Mac1. These results are consistent with the C1 motif being the copper-regulatory switch. Both cysteine-rich motifs exhibited transactivation activity, although the C1 activator was weak relative to the C2 activator. Limited copper metalloregulation of Mac1 was observed with only the C1 activator fused to the N-terminal DNA binding domain. Thus, the two Cys-rich motifs appear to function independently. The C1 motif appears to be a functional copper-regulatory domain.
Asunto(s)
Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Cisteína , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/metabolismo , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
The Zap1 transcriptional activator of Saccharomyces cerevisiae plays a major role in zinc homeostasis by inducing the expression of several genes under zinc-limited growth conditions. This activation of gene expression is mediated by binding of the protein to one or more zinc-responsive elements present in the promoters of its target genes. To better understand how Zap1 functions, we mapped its DNA binding domain using a combined in vivo and in vitro approach. Our results show that the Zap1 DNA binding domain maps to the carboxyl-terminal 194 amino acids of the protein; this region contains five of its seven potential zinc finger domains. Fusing this region to the Gal4 activation domain complemented a zap1Delta mutation for low zinc growth and also conferred high level expression on a zinc-responsive element-lacZ reporter. In vitro, the purified 194-residue fragment bound to DNA with a high affinity (dissociation constant in the low nanomolar range) similar to that of longer fragments of Zap1. Furthermore, by deletion and site-directed mutagenesis, we demonstrated that each of the five carboxyl-terminal zinc fingers are required for high affinity DNA binding.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Transactivadores/genética , Activación Transcripcional , Dedos de Zinc/genética , Zinc/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Expresión Génica , Genes Reporteros , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Saccharomyces cerevisiae , Transactivadores/química , Factores de Transcripción/genéticaRESUMEN
Mac1 is a metalloregulatory protein that regulates expression of the high affinity copper transport system in the yeast Saccharomyces cerevisiae. Under conditions of high copper concentration, Mac1 represses transcription of genes coding for copper transport proteins. Mac1 binds to DNA sequences called copper response elements (CuREs), which have the consensus sequence 5'-TTTGC(T/G)C(A/G)-3'. Mac1 contains two zinc binding sites, a copper binding site, and the sequence motif RGRP, which has been found in other proteins to mediate binding to the minor groove of A/T-rich sequences in DNA. We have used hydroxyl radical footprinting, missing nucleoside, and methylation interference experiments to investigate the structure of the complex of the DNA binding domain of Mac1 (called here Mac1(t)) with the two CuRE sites found in the yeast CTR1 promoter. We conclude from these experiments that Mac1(t) binds in a modular fashion to DNA, with its RGRP AT-hook motif interacting with the TTT sequence at the 5' end of the CTR1 CuRE site, and with another DNA-binding module(s) binding in the adjacent major groove in the GCTCA sequence.
Asunto(s)
Proteínas de Transporte de Catión , ADN de Hongos/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transportador de Cobre 1 , Proteínas Fúngicas/genética , Radical Hidroxilo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras GenéticasRESUMEN
Human neuronal growth inhibitory factor (GIF), a metallothionein-like protein classified as metallothionein-3, impairs the survival and the neurite formation of cultured neurons. Despite its approximately 70% amino acid sequence identity with those of mammalian metallothioneins (MT-1 and MT-2 isoforms), only GIF exhibits growth inhibitory activity. In this study, structural features of the metal-thiolate clusters in recombinant Zn(7)- and Cd(7)-GIF, and in part also in synthetic GIF (68 amino acids), were investigated by using circular dichroism (CD) and (113)Cd NMR. The CD and (113)Cd NMR studies of recombinant Me(7)-GIF confirmed the existence of distinct Me(4)S(11)- and Me(3)S(9)-clusters located in the alpha- and beta-domains of the protein, respectively. Moreover, a mutual structural stabilization of both domains was demonstrated. The (113)Cd NMR studies of recombinant (113)Cd(7)-GIF were conducted at different magnetic fields (66.66 and 133.33 MHz) and temperatures (298 and 323 K). At 298 K the spectra revealed seven (113)Cd signals at 676, 664, 651, 644, 624, 622, and 595 ppm. A striking feature of all resonances is the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz), which account for the absence of cross-peaks in [(113)Cd, (113)Cd] COSY. On the basis of a close correspondence in chemical shift positions of the (113)Cd signals at 676, 624, 622, and 595 ppm with those obtained in our previous studies of (113)Cd(4)-GIF(32-68) [Hasler, D. W., Faller, P., and Vasák, M. (1998) Biochemistry 37, 14966], these resonances can be assigned to a Cd(4)S(11)-cluster in the alpha-domain of (113)Cd(7)-GIF. Consequently, the remaining three (113)Cd signals at 664, 651, and 644 ppm originate from a Me(3)S(9) cluster in the beta-domain. However, the latter resonances show a markedly reduced and temperature-independent intensity (approximately 20%) when compared with those of the alpha-domain, indicating that the majority of the signal intensity remained undetected. To account for the observed NMR features of (113)Cd(7)-GIF, we suggest that dynamic processes acting on two different NMR time scales are present: (i) fast exchange processes among conformational cluster substates giving rise to broad, weight-averaged resonances and (ii) additional very slow exchange processes within the beta-domain associated with the formation of configurational cluster substates. The implications of the structure fluctuation for the biological activity of GIF are discussed.
Asunto(s)
Inhibidores de Crecimiento/química , Metalotioneína/química , Metales Pesados/química , Proteínas del Tejido Nervioso/química , Secuencia de Aminoácidos , Cadmio/química , Dicroismo Circular , Inhibidores de Crecimiento/síntesis química , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/metabolismo , Humanos , Isótopos , Metalotioneína/metabolismo , Metalotioneína 3 , Metales Pesados/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/síntesis química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Análisis Espectral , Termodinámica , Zinc/químicaAsunto(s)
Proteínas de Transporte de Catión , Cobre/metabolismo , Regulación de la Expresión Génica , Proteínas de Plantas , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Proteínas Nucleares/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
Mac1 mediates copper (Cu)-dependent expression of genes involved in high-affinity uptake of copper ions in Saccharomyces cerevisiae. Mac1 is a transcriptional activator in Cu-deficient cells, but is inhibited in Cu-replete cells. Mac1 resides within the nucleus in both Cu-deficient and Cu-loaded cells. Cu inhibition of Mac1 appears to result from binding of eight copper ions within a C-terminal segment consisting of two Cys-rich motifs. In addition, two zinc ions are bound within the N-terminal DNA-binding domain. Only 4-5 mol. eq. Cu are bound to a mutant Mac1 (His279Gln substitution) that is impervious to Cu inhibition. The CuMac1 complex is luminescent, indicative of copper bound in the Cu(I) state. Cu binding induces a molecular switch resulting in an intramolecular interaction in Mac1 between the N-terminal DNA-binding domain and the C-terminal activation domain. This allosteric interaction is Cu dependent and is not observed when Mac1 contained the mutant His279Gln substitution. Fusion of the minimal DNA-binding domain of Mac1 (residues 1-159) to the minimal Cu-binding activation domain (residues 252-341) yields a functional Cu-regulated transcriptional activator. These results suggest that Cu repression of Mac1 arises from a Cu-induced intramolecular interaction that inhibits both DNA binding and transactivation activities.
Asunto(s)
Cobre/farmacología , Proteínas Nucleares/química , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/química , Factores de Transcripción/química , Sustitución de Aminoácidos , Cobre/metabolismo , Cisteína , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiae/genética , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
The high affinity uptake systems for iron and copper ions in Saccharomyces cerevisiae involve metal-specific permeases and two known cell surface Cu(II) and Fe(III) metalloreductases, Fre1 and Fre2. Five novel genes found in the S. cerevisiae genome exhibit marked sequence similarity to Fre1 and Fre2, suggesting that the homologs are part of a family of proteins related to Fre1 and Fre2. The homologs are expressed genes in S. cerevisiae, and their expression is metalloregulated as is true with FRE1 and FRE2. Four of the homologs (FRE3-FRE6) are specifically iron-regulated through the Aft1 transcription factor. These genes are expressed either in cells limited for iron ion uptake by treatment with a chelator or in cells lacking the high affinity iron uptake system. Expression of FRE3-FRE6 is elevated in AFT1-1 cells and attenuated in aft1 null cells, showing that iron modulation occurs through the Aft1 transcriptional activator. The fifth homolog FRE7 is specifically copper-metalloregulated. FRE7 is expressed in cells limited in copper ion uptake by a Cu(I)-specific chelator or in cells lacking the high affinity Cu(I) permeases. The constitutive expression of FRE7 in MAC1 cells and the lack of expression in mac1-1 cells are consistent with Mac1 being the critical transcriptional activator of FRE7 expression. The 5' promoter sequence of FRE7 contains three copper-responsive promoter elements. Two elements are critical for Mac1-dependent FRE7 expression. Combinations of either the distal and central elements or the central and proximal elements result in copper-regulated FRE7 expression. Spacing between Mac1-responsive sites is important as shown by the attenuated expression of FRE7 and CTR1 when two elements are separated by over 100 base pairs. From the three Mac1-responsive elements in FRE7, a new consensus sequence for Mac1 binding can be established as TTTGC(T/G)C(A/G).
Asunto(s)
Cobre/metabolismo , FMN Reductasa , Hierro/metabolismo , NADH NADPH Oxidorreductasas/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Calmodulina/genética , Clonación Molecular , Genotipo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADH NADPH Oxidorreductasas/biosíntesis , NADH NADPH Oxidorreductasas/química , Oligodesoxirribonucleótidos , Regiones Promotoras Genéticas , Conformación Proteica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
Mac1 from Saccharomyces cerevisiae activates transcription of genes, including CTR1 in copper-deficient cells. N-terminal fusions of Mac1 with the herpes simplex VP16 activation domain were used to show that residues 1-159 in Mac1 constitute the minimal DNA binding domain. Mac1-(1-159) purified from Escherichia coli contains two bound Zn(II) ions. Electrophoretic mobility shift assays showed direct and specific binding by Mac1-(1-159) to a DNA duplex containing the copper-responsive element TTTGCTCA. The DNA binding affinity of Mac1-(1-159) for a duplex containing a single promoter element or an inverted repeat was 5 nM for the 1:1 complex. The N-terminal 40-residue segment of Mac1 is homologous to the DNA binding zinc module found in the copper-activated transcription factors Ace1 and Amt1. A MAC1 mutation yielding a Cys11 --> Tyr substitution at the first candidate zinc ligand position relative to Ace1 resulted in a loss of in vivo function. Two TTTGCTCA promoter elements are necessary for efficient Mac1-mediated transcriptional activation. The elements appear to function synergistically. Increasing the number of elements yields more than additive enhancements in CTR1 expression.
Asunto(s)
Cobre/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Cisteína , Escherichia coli , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/genética , Oligodesoxirribonucleótidos , Sondas de Oligonucleótidos , Regiones Promotoras Genéticas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de Transcripción/química , Factores de Transcripción/genética , Tirosina , Zinc/metabolismoRESUMEN
The three dimensional structure of the N-terminal domain (residues 1-42) of the copper-responsive transcription factor Amtl from Candida glabrata has been determined by two-dimensional 1H-correlated nuclear magnetic resonance (NMR) methods. The domain contains an array of zinc-binding residues (Cys-X2-Cys-X8-Cys-X-His) that is conserved among a family of Cu-responsive transcription factors. The structure is unlike those of previously characterized zinc finger motifs, and consists of a three-stranded antiparallel beta-sheet with two short helical segments that project from one end of the beta-sheet. Conserved residues at positions 16, 18 and 19 form a basic patch that may be important for DNA binding.
Asunto(s)
Candida/química , Proteínas de Unión al ADN/química , Estructura Secundaria de Proteína , Factores de Transcripción/química , Zinc/química , Secuencia de Aminoácidos , Cobre/farmacología , Cisteína/química , Proteínas Fúngicas , Datos de Secuencia Molecular , Resonancia Magnética Nuclear BiomolecularRESUMEN
Metal-responsive transcription factors exist in yeast to modulate expression of genes that encode proteins involved in cellular uptake of copper, iron and zinc ions. These signal transduction pathways function in the cellular regulation of the intracellular concentration of free metal ions. A second component of metal homeostasis is the regulation of metal-ion binding through protein-mediated metallation. Copper-specific chaperones exist in yeast that route copper ions to the site of biosynthesis of copper-metalloenzymes.
Asunto(s)
Regulación Fúngica de la Expresión Génica/genética , Metales/metabolismo , Saccharomyces cerevisiae/fisiología , Cobre/metabolismo , Proteínas de Unión al ADN/química , Hierro/metabolismo , Metaloproteínas/metabolismo , Factores de Transcripción/genética , Zinc/metabolismoRESUMEN
Assembly of functional cytochrome oxidase in yeast requires Cox17, which has been postulated to deliver copper ions to the mitochondrion for insertion into the enzyme. This role for Cox17 is supported by the observation that it binds copper as a binuclear cuprous-thiolate cluster. X-ray absorption spectroscopy, together with UV-visible absorption and emission spectroscopy, indicates the presence of bound cuprous ions, trigonally coordinated by thiolate ligands. Analysis of the EXAFS shows three Cu-S bonds at 2.26 A, plus a short Cu-Cu distance of 2.7 A, indicating a binuclear cluster in Cox17. The cuprous-thiolate cluster in Cox17 is substantially more labile than structurally related clusters in metallothioneins.