Background: The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu⁺ or Cd²⁺. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion. Results: HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd²⁺, Zn²⁺ or Cu²⁺ and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd₆-HpCdMT and Cu₁₂-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions. Conclusion: The Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

Summary: Zinc is an essential metal that, when in excess, can be deleterious to the cell. Therefore, homeostatic mechanisms for this cation must be finely tuned. To better understand the response of yeast in front of an excess of zinc, we screened a systematic deletion mutant library for altered growth in the presence of 6 mM zinc. Eighty‐nine mutants exhibited increased zinc sensitivity, including many genes involved in vacuolar assembling and biogenesis. Interestingly, a mutant lacking the Aft1 transcription factor, required for the transcriptional response to iron starvation, was found to be highly sensitive to zinc. Genome‐wide transcriptional profiling revealed that exposure to 5 mM ZnCl₂ results in rapid increase in the expression of numerous chaperones required for proper protein folding or targeting to vacuole and mitochondria, as well as genes involved in stress response (mainly oxidative), sulphur metabolism and some components of the iron regulon. The effect of the lack of Aft1 both in the absence and in the presence of zinc overload was also investigated. Exposure to high zinc generated reactive oxygen species and markedly decreased glutathione content. Interestingly, zinc excess results in decreased intracellular iron content and aconitase and cytochrome c activities in stationary‐phase cultures. These findings suggest that high zinc levels may alter the assembly and/or function of iron–sulphur‐containing proteins, as well as the biosynthesis of haem groups, thus establishing a link between zinc, iron and sulphur metabolism.

Summary: Crs5 is a Saccharomyces cerevisiae Metallothionein (MT), non‐homologous to the paradigmatic Cu‐thionein Cup1. Although considered a secondary copper‐resistance agent, we show here that it determines survival under zinc overload in a CUP1‐null background. Its overexpression prevents the deleterious effects exhibited by CUP1‐CRS5‐null cells when exposed to combined Zn/Cu, as it does the mouse MT1 Zn‐thionein, but not Cup1. The detailed characterization of Crs5 in vivo and in vitro Zn(II)‐, Cd(II)‐ and Cu(I)‐binding abilities fully supports its resemblance to mammalian MTs. Hence, Crs5 exhibits a good divalent metal‐binding ability, yielding homometallic, highly chiral and stable Zn and Cd complexes when expressed in media enriched with these metal ions. In Cu‐supplemented cultures, heterometallic Zn,Cu complexes are recovered, unless aeration is kept to a minimum. These features define a Crs5 dual metal‐binding behaviour that is significantly closer to Zn‐thioneins than to Cu‐thioneins. Protein sequence similarities fully support these findings. Overall, a Crs5 function in global metal cell homeostasis, based on its Zn‐binding features, is glimpsed. The comparative evaluation of Crs5 in the framework of MT functional differentiation and evolution allows its consideration as a representative of the primeval eukaryotic forms that progressively evolved to give rise to the Zn‐thionein lineage.