A chemical potentiator of copper-accumulation used to investigate the iron-regulons of Saccharomyces cerevisiae.

The extreme resistance of Saccharomyces cerevisiae to copper is overcome by 2-(6-benzyl-2-pyridyl)quinazoline (BPQ), providing a chemical-biology tool which has been exploited in two lines of discovery. First, BPQ is shown to form a red (BPQ)2 Cu(I) complex and promote Ctr1-independent copper-accumulation in whole cells and in mitochondria isolated from treated cells. Multiple phenotypes, including loss of aconitase activity, are consistent with copper-BPQ mediated damage to mitochondrial iron-sulphur clusters. Thus, a biochemical basis of copper-toxicity in S. cerevisiae is analogous to other organisms. Second, iron regulons controlled by Aft1/2, Cth2 and Yap5 that respond to mitochondrial iron-sulphur cluster status are modulated by copper-BPQ causing iron hyper-accumulation via upregulated iron-import. Comparison of copper-BPQ treated, untreated and copper-only treated wild-type and fra2Δ by RNA-seq has uncovered a new candidate Aft1 target-gene (LSO1) and paralogous non-target (LSO2), plus nine putative Cth2 target-transcripts. Two lines of evidence confirm that Fra2 dominates basal repression of the Aft1/2 regulons in iron-replete cultures. Fra2-independent control of these regulons is also observed but CTH2 itself appears to be atypically Fra2-dependent. However, control of Cth2-target transcripts which is independent of CTH2 transcript abundance or of Fra2, is also quantified. Use of copper-BPQ supports a substantial contribution of metabolite repression to iron-regulation.

Cyanobacterial metallochaperone inhibits deleterious side reactions of copper.

Copper metallochaperones supply copper to cupro-proteins through copper-mediated protein-protein-interactions and it has been hypothesized that metallochaperones thereby inhibit copper from causing damage en route. Evidence is presented in support of this latter role for cyanobacterial metallochaperone, Atx1. In cyanobacteria Atx1 contributes towards the supply of copper to plastocyanin inside thylakoids but it is shown here that in copper-replete medium, copper can reach plastocyanin without Atx1. Unlike metallochaperone-independent copper-supply to superoxide dismutase in eukaryotes, glutathione is not essential for Atx1-independent supply to plastocyanin: Double mutants missing atx1 and gshB (encoding glutathione synthetase) accumulate the same number of atoms of copper per cell in the plastocyanin pool as wild type. Critically, Δatx1ΔgshB are hypersensitive to elevated copper relative to wild type cells and also relative to ΔgshB single mutants with evidence that hypersensitivity arises due to the mislocation of copper to sites for other metals including iron and zinc. The zinc site on the amino-terminal domain (ZiaA(N)) of the P(1)-type zinc-transporting ATPase is especially similar to the copper site of the Atx1 target PacS(N), and ZiaA(N) will bind Cu(I) more tightly than zinc. An NMR model of a substituted-ZiaA(N)-Cu(I)-Atx1 heterodimer has been generated making it possible to visualize a juxtaposition of residues surrounding the ZiaA(N) zinc site, including Asp(18), which normally repulse Atx1. Equivalent repulsion between bacterial copper metallochaperones and the amino-terminal regions of P(1)-type ATPases for metals other than Cu(I) is conserved, again consistent with a role for copper metallochaperones to withhold copper from binding sites for other metals.

Structure and metal loading of a soluble periplasm cuproprotein.

A copper-trafficking pathway was found to enable Cu(2+) occupancy of a soluble periplasm protein, CucA, even when competing Zn(2+) is abundant in the periplasm. Here, we solved the structure of CucA (a new cupin) and found that binding of Cu(2+), but not Zn(2+), quenches the fluorescence of Trp(165), which is adjacent to the metal site. Using this fluorescence probe, we established that CucA becomes partly occupied by Zn(2+) following exposure to equimolar Zn(2+) and Cu(2+). Cu(2+)-CucA is more thermodynamically stable than Zn(2+)-CucA but k((Zn→Cu)exchange) is slow, raising questions about how the periplasm contains solely the Cu(2+) form. We discovered that a copper-trafficking pathway involving two copper transporters (CtaA and PacS) and a metallochaperone (Atx1) is obligatory for Cu(2+)-CucA to accumulate in the periplasm. There was negligible CucA protein in the periplasm of ΔctaA cells, but the abundance of cucA transcripts was unaltered. Crucially, ΔctaA cells overaccumulate low M(r) copper complexes in the periplasm, and purified apoCucA can readily acquire Cu(2+) from ΔctaA periplasm extracts, but in vivo apoCucA fails to come into contact with these periplasmic copper pools. Instead, copper traffics via a cytoplasmic pathway that is coupled to CucA translocation to the periplasm.

Interaction between cyanobacterial copper chaperone Atx1 and zinc homeostasis.

Cyanobacterial Atx1 is a copper chaperone which interacts with two copper-transporting ATPases to assist copper supply to plastocyanin and cytochrome oxidase. ZiaA is a Zn(2+)-exporting ATPase and ziaA expression is regulated by ZiaR. Here we show that gene expression from the ziaA operator promoter, monitored using reverse transcriptase PCR and lacZ fusions, is elevated in Deltaatx1 mutants. Although Cu(+) tightly binds recombinant ZiaR in vitro, Cu(+) is less effective at dissociating ZiaR-DNA complexes than Zn(2+) and crucially ziaA expression responds to Zn(2+) but not copper in both wild-type and Deltaatx1 cells. Consistent with enhanced expression of ZiaA, Deltaatx1 cells have slightly elevated Zn(2+) resistance. Recombinant Zn(2+)-Atx1 is recovered from Zn(2+)-supplemented Escherichia coli and even after copper supplementation substantial amounts of Zn(2+)-Atx1 are isolated. Taken together, these data suggest that Zn(2+)-Atx1 can form in vivo.

Response of Schizosaccharomyces pombe to zinc deficiency.

A component of the cellular response to zinc deficiency operates via control of transcript abundance. Therefore, microarray analysis was employed to identify Schizosaccharomyces pombe genes whose mRNA levels are regulated by intracellular zinc status. A set of 57 genes whose mRNA levels were substantially reduced in response to zinc deficiency was identified, while the mRNA levels of 63 genes were increased by this condition. In order to investigate the mechanisms that control these responses, a genetic screen was employed to identify mutants with defective zinc-responsive gene expression. Two strains (II-1 and V7) that were identified by this screen harbor mutations that are linked to zrt1+, which encodes a putative Zrt/IRT-like protein (ZIP) zinc uptake transporter. Importantly, zrt1+ mRNA levels are increased in response to zinc deprivation, and cells lacking functional Zrt1 are highly impaired in their ability to proliferate at limiting zinc concentrations. Furthermore, zrt1 null cells were found to have severely reduced zinc contents, indicating that Zrt1 functions as a key regulator of intracellular zinc levels in fission yeast. The deletion of fet4+, another zinc-responsive gene encoding a putative metal ion transporter, exacerbated the phenotypes associated with the loss of Zrt1, suggesting that Fet4 also plays a role in zinc uptake under limiting conditions.

Co(ll)-detection does not follow Kco(ll) gradient: channelling in Co(ll)-sensing.

The MerR-like transcriptional activator CoaR detects surplus Co(ll) to regulate Co(ll) efflux in a cyanobacterium. This organism also has cytosolic metal-sensors from three further families represented by Zn(ll)-sensors ZiaR and Zur plus Ni(ll)-sensor InrS. Here we discover by competition with Fura-2 that CoaR has KCo(ll) weaker than 7 × 10(-8) M, which is weaker than ZiaR, Zur and InrS (KCo(ll) = 6.94 ± 1.3 × 10(-10) M; 4.56 ± 0.16 × 10(-10) M; and 7.69 ± 1.1 × 10(-9) M respectively). KCo(ll) for CoaR is also weak in the CoaR-DNA adduct. Further, Co(ll) promotes DNA-dissociation by ZiaR and DNA-association by Zur in vitro in a manner analogous to Zn(ll), as monitored by fluorescence anisotropy. After 48 h exposure to maximum non-inhibitory [Co(ll)], CoaR responds in vivo yet the two Zn(ll)-sensors do not, despite their tighter KCo(ll) and despite Co(ll) triggering allostery in ZiaR and Zur in vitro. These data imply that the two Zn(ll) sensors fail to respond because they fail to gain access to Co(ll) under these conditions in vivo. Several lines of evidence suggest that CoaR is membrane associated via a domain with sequence similarity to precorrin isomerase, an enzyme of vitamin B12 biosynthesis. Moreover, site directed mutagenesis reveals that transcriptional activation requires CoaR residues that are predicted to form hydrogen bonds to a tetrapyrrole. The Co(ll)-requiring vitamin B12 biosynthetic pathway is also membrane associated suggesting putative mechanisms by which Co(ll)-containing tetrapyrroles and/or Co(ll) ions are channelled to CoaR.