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 aft1Δaft2Δ deletion was generated. Growth assays established that the single aft2Δ strain exhibited no iron-dependent phenotype. However, the double-mutant aft1Δaft2Δ strain was more sensitive to low-iron growth conditions than the single-mutant aft1Δ strain. A mutant allele of AFT2 (AFT2-1up), or overexpression of the wild-type AFT2 gene, led to partial complementation of the respiratory-deficient phenotype of the aft1Δ strain. The AFT2-1upallele also increased the uptake of59Fe in an aft1Δ 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, AFT2-1up-dependent activation was independent of Aft1p. The kinetics of induction of two genes activated by the AFT2-1upallele 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.
Cytochrome c oxidase (CcO) is an oligomeric complex localized within the mitochondrial inner membrane. Assembly of the active oxidase complex requires the coordinate assembly of subunits synthesized ...in both the cytoplasm and the mitochondrion. In addition, assembly is dependent on the insertion of five types of cofactors, including two hemes, three copper ions, and one Zn, Mg, and Na ion. A series of accessory proteins are critical for synthesis of the heme A cofactor and insertion of the copper ions. This Account will focus on the steps in the coordinate assembly of CcO subunits, the formation of heme A, and the delivery and insertion of copper ions.
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.
The assembly of the copper sites in cytochrome c oxidase involves a series of accessory proteins, including Cox11, Cox17, and Sco1. The two mitochondrial inner membrane proteins
Cox11 and Sco1 are ...thought to be copper donors to the Cu B and Cu A sites of cytochrome oxidase, respectively, whereas Cox17 is believed to be the copper donor to Sco1 within the intermembrane
space. In this report we show Cox17 is a specific copper donor to both Sco1 and Cox11. Using in vitro studies with purified proteins, we demonstrate direct copper transfer from CuCox17 to Sco1 or Cox11. The transfer is specific
because no transfer occurs to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, a
C57Y mutant of Cox17 fails to transfer copper to Sco1 but is competent for copper transfer to Cox11. The in vitro transfer studies were corroborated by a yeast cytoplasm expression system. Soluble domains of Sco1 and Cox11, lacking the
mitochondrial targeting sequence and transmembrane domains, were expressed in the yeast cytoplasm. Metallation of these domains
was strictly dependent on the co-expression of Cox17. Thus, Cox17 represents a novel copper chaperone that delivers copper
to two proteins.
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 μM 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.
The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known mitochondrial
cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase ...(CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1. Only a small fraction of mitochondrial
copper is associated with these cuproproteins. The bulk of the remainder is localized within the matrix as a soluble, anionic,
low molecular weight complex. The identity of the matrix copper ligand is unknown, but the bulk of the matrix copper fraction
is not protein-bound. The mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition
of copper salts to the growth medium leads to an increase in mitochondrial copper, yet the expansion of this matrix pool does
not induce any respiration defects. The matrix copper pool is accessible to a heterologous cuproenzyme. Co-localization of
human Sod1 and the metallochaperone CCS within the mitochondrial matrix results in suppression of growth defects of sod2 Î cells. However, in the absence of CCS within the matrix, the activation of human Sod1 can be achieved by the addition of
copper salts to the growth medium.
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 Zn7-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 113Cd NMR spectroscopy, the structural features of the metal−thiolate clusters in the double mutant Cd7-mutMT-3 were investigated and compared with those of wild-type Cd7-MT-3 Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vašák, M. (1999) Biochemistry 38, 10158 and the well characterized Cd7-MT-2a from rabbit liver. Similarly to 113Cd7-MT-3 the 113Cd NMR spectrum of 113Cd7-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 Cd4S11 cluster in the α-domain and a Cd3S9 cluster in the β-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 113Cd−113Cd couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in 113Cd7-mutMT-3 the temperature rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd3S9 cluster resonances, no such temperature effect was observed in 113Cd7-MT-3. To account for the observed NMR features, a dynamic structural model for the β-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 113Cd7-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 β-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.
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 γ-lyase encoded by CYS3. Several genes that are differentially expressed in cells containing a constitutively active Mac1, designated Mac1up1, 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 andCRS5 and two genes, FET3 and FTR1, in the iron uptake system. Copper-induced FET3 andFTR1 expression arises from an indirect copper effect on cellular iron pools.
Saccharomyces cerevisiae responds to iron deprivation by increased transcription of the iron regulon, including the high affinity cell-surface transport
system encoded by FET3 and FTR1 . Here we ...demonstrate that transcription of these genes does not respond directly to cytosolic iron but rather to the mitochondrial
utilization of iron for the synthesis of iron-sulfur (Fe-S) clusters. We took advantage of a mutant form of an iron-dependent
enzyme in the sterol pathway (Erg25-2p) to assess cytosolic iron levels. We showed that disruption of mitochondrial Fe-S biosynthesis,
which results in excessive mitochondrial iron accumulation, leads to transcription of the iron transport system independent
of the cytosolic iron level. There is an inverse correlation between the activity of the mitochondrial Fe-S-containing enzyme
aconitase and the induction of FET3 . Regulation of transcription by Fe-S biosynthesis represents a mechanism by which cellular iron acquisition is integrated
with mitochondrial iron metabolism.
The transcription factors Aft1p and Aft2p from Saccharomyces cerevisiae regulate the expression of genes that are involved in iron homeostasis. In vitro studies have shown that both transcription ...factors bind to an iron-responsive element (FeRE) that is present in the upstream region of genes in the iron regulon. We have used DNA microarrays to distinguish the genes that are activated by Aft1p and Aft2p and to establish for the first time that each factor gives rise to a unique transcriptional profile due to the differential expression of individual iron-regulated genes. We also show that both Aft1p and Aft2p mediate the in vivo expression of FET3 and FIT3 through a consensus FeRE. In addition, both proteins regulate MRS4 via a variant FeRE with Aft2p being the stronger activator from this particular element. Like other paralogous pairs of transcription factors within S. cerevisiae, Aft1p and Aft2p are able to interact with the same promoter elements while maintaining specificity of gene activation.
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