Copper is an essential yet toxic trace element. The Ctr1 family of proteins plays a critical role for copper uptake in eukaryotes. However, the mechanisms of action of Ctr1 are largely unknown. Our previous data demonstrated that copper transport induces conformational changes in the cytosolic C terminus of the yeast Saccharomyces cerevisiae Ctr1. To define the physiological significance of this molecular event and gain better insights into the mechanism of Ctr1-mediated copper uptake, we have characterized the functional roles of the Ctr1 C terminus. A Ctr1 mutant lacking the entire C-terminal cytosolic tail is functional in high affinity copper uptake; however, yeast cells expressing this mutant are extremely sensitive to excess copper. Toxic copper uptake is not attributed to elevated expression or distinct subcellular localization of this mutant as compared with wild type Ctr1. Further characterization of the function of Ctr1 containing deletions or site-directed mutations at the C terminus indicates a structural role for the C terminus in controlling Ctr1 activities. In response to excess copper, Ctr1-mediated copper transport is rapidly blocked in a C terminus-dependent mechanism associated with direct binding of copper. We propose that conformational changes in the cytosolic tail of yeast Ctr1 by copper sensing within this domain lead to the inhibition of Ctr1-mediated copper transport. These data suggest a new regulatory mechanism by which yeast cells maintain homeostatic copper acquisition.

Fu brick tea (FBT) is one of the major brands of dark tea. Microbial fermentation is considered the key step in the development of the special characteristics of FBT. The systemic corelationship of the microbiome and metabolomics during manufacture of Fu brick tea is not fully understood. In this study, we comprehensively explored the microbiome and metabolite dynamic evolution during the FBT manufacturing processes, and revealed decisive factors for the quality and safety of FBT based on the grouped methods of metabolomics combined with biochemical measurements, microbiome sequencing combined with quantitative polymerase chain reaction (PCR), and multiplex analysis. Both the microbiome and quantitative PCR showed that fungi displayed concentrated distribution characteristics in the primary dark tea samples, while bacterial richness increased during the flowering processes and ripening period. All microorganism species, as well as dominant fungi and bacteria, were identified in the distinct processes periods. A total of 178 metabolites were identified, and 34 of them were characterized as critical metabolites responsible for metabolic changes caused by the corresponding processes. Metabolic analysis showed that most metabolites were decreased during the FBT manufacturing processes, with the exception of gallic acid. Multivariate analysis verified that the critical metabolites were correlated with specific dominant microbial species. All the top fungal species except unclassified_g_ Aspergillus showed positive correlations with six critical metabolites (L-The, epigallocatechin (EGC), Gln, tea polyphenol (TP), tea polysaccharides (TPs) and caffeine). Five of the top bacteria species (Cronobacter, Klebsiella, Pantoea, Pluralibacter, and unclassified_ f_Entero-bacteriaceae) showed positive correlations with epigallocatechins and tea polyphenols, while the other 11 top bacterial species correlated negatively with all the critical metabolites. The content of amino acids, tea polyphenols, tea polysaccharides, and flavonoids was reduced during microbial fermentation. In conclusion, our results reveal that microbial composition is the critical factor in changing the metabolic profile of FBT. This discovery provides a theoretical basis for improving the quality of FBT and enhancing its safety.

Acquisition and detoxification of metal ions are vital biological processes. Given the requirement of metallochaperones in cellular copper distribution and metallation of cuproproteins, this study investigates whether the metallochaperones also deliver metal ions for transporters functioning in metal detoxification. Resistance to excess cadmium and copper of the yeast Saccharomyces cerevisiae, which is conferred by PCA1 and CaCRP1 metal efflux P-type ATPases, respectively, does not rely on known metallochaperones, Atx1p, Ccs1p, and Cox17p. Copper deficiency induced by the expression of CaCRP1 encoding a copper exporter occurs in the absence of Atx1p. Intriguingly, CCS1 encoding the copper chaperone for superoxide dismutase 1 (Sod1p) is necessary for cadmium resistance that is mediated by Ycf1p, a vacuolar cadmium sequestration transporter. This is attributed to Ccs1p's role in the maturation of Sod1p rather than its direct interaction with Ycf1p for cadmium transfer. Functional defect in Ycf1p associated with the absence of Sod1p as well as another antioxidant enzyme Glr1p is rescued by anaerobic growth or substitutions of specific cysteine residues of Ycf1p to alanine or serine. This further supports oxidative inactivation of Ycf1p in the absence of Ccs1p, Sod1p, or Glr1p. These results provide new insights into the mechanisms of metal metabolism, interaction among metal ions, and the roles for antioxidant systems in metal detoxification. Copper metabolism and antioxidant enzymes maintain the function of Ycf1p for cadmium defense.

Acquisition and distribution of metal ions support a number of biological processes. Here we show that respiratory growth of and iron acquisition by the yeast Saccharomyces cerevisiae relies on potassium (K⁺) compartmentalization to the trans-Golgi network via Kha1p, a K⁺/H⁺ exchanger. K⁺ in the trans-Golgi network facilitates binding of copper to the Fet3p multi-copper ferroxidase. The effect of K⁺ is not dependent on stable binding with Fet3p or alteration of the characteristics of the secretory pathway. The data suggest that K⁺ acts as a chemical factor in Fet3p maturation, a role similar to that of cations in folding of nucleic acids. Up-regulation of KHA1 gene in response to iron limitation via iron-specific transcription factors indicates that K⁺ compartmentalization is linked to cellular iron homeostasis. Our study reveals a novel functional role of K⁺ in the binding of copper to apoFet3p and identifies a K⁺/H⁺ exchanger at the secretory pathway as a new molecular factor associated with iron uptake in yeast.