Abstract
In fission yeast, the genes encoding proteins that are components of the copper transporter family are controlled at the transcriptional level by the Cuf1 transcription factor. Under low copper availability, Cuf1 induces expression of the copper transporter genes. In contrast, sufficient levels of copper inactivate Cuf1 and expression of its target genes. Our study reveals that Cuf1 harbors a putative copper-binding motif, Cys-X-Cys-X(3)-Cys-X-Cys-X(2)-Cys-X(2)-His, within its carboxyl-terminal region to sense changing environmental copper levels. Binding studies reveal that the amino-terminal 174-residue segment of Cuf1 expressed as a fusion protein in Escherichia coli specifically interacts with the cis-acting copper transporter promoter element CuSE (copper-signaling element). Within this region, the first 61 amino acids of Cuf1 exhibit more overall homology to the Saccharomyces cerevisiae Ace1 copper-detoxifying factor (from residues 1 to 63) than to Mac1, its functional ortholog. Consistently, we demonstrate that a chimeric Cuf1 protein bearing the amino-terminal 63-residue segment of Ace1 complements cuf1 Delta null phenotypes. Furthermore, we show that Schizosaccharomyces pombe cuf1Delta mutant cells expressing the full-length S. cerevisiae Ace1 protein are hypersensitive to copper ions, with a concomitant up-regulation of CuSE-mediated gene expression in fission yeast. Taken together, these studies reveal that S. cerevisiae Ace1 1-63 is functionally exchangeable with S. pombe Cuf1 1-61, and the nature of the amino acids located downstream of this amino-terminal conserved region may be crucial in dictating the type of regulatory response required to establish and maintain copper homeostasis.
Highlights
Copper is an essential element for aerobic life [1]
Our study reveals that Cuf1 harbors a putative copper-binding motif, Cys-X-Cys-X3Cys-X-Cys-X2-Cys-X2-His, within its carboxyl-terminal region to sense changing environmental copper levels
To detect and respond to elevated environmental copper levels, S. cerevisiae cells possess the Ace1 copper-detoxifying transcription factor that can prevent the accumulation of copper to toxic concentrations by trans-activating the expression of the copper-sequestering CUP1 and CRS5 metallothionein (MT) genes, and the copper, zinc-SOD1 gene [11,12,13,14,15,16,17,18]
Summary
Yeast Strains and Culture Conditions—The S. pombe strains used in this study were the wild type FY435 (hϩ his366 leu ura4-⌬18 ade6-M210) [52] and the cuf1⌬ disruption strain JSY17 (hϩ his366 leu ura4-⌬18 ade6-M210 cuf1⌬::ura4ϩ), unless otherwise stated. To create the cuf mutant allele M7, a linker containing an XbaI site was inserted inframe within the cuf1ϩ gene at position ϩ369 relative to the first nucleotide of the initiator codon. A 380-bp PstI-XbaI fragment of the cuf1ϩ gene up to ϩ372 from the start codon (residues 1–124) was isolated by PCR from pSPcuf1ϩS-B/4.2. 107, Cys-108, Cys-115, and Cys-116 to tyrosine residues) was swapped for an identical DNA region into the pSPcuf M3 S-B/4.2 plasmid (instead of pSPcuf1ϩS-B/4.2), creating the cuf mutant allele M8. The pSK cuf1⌬175–300 plasmid was digested with PstI and BamHI and cloned into the corresponding sites of pSPcuf1ϩS-B/4.2, generating a plasmid bearing a deletion of 125 codons within the middle region of cuf1ϩ. To create a chimeric plasmid that has the first codons of ACE1 fused to cuf1ϩ through 411, an SmaI site was engineered by PCR mutagenesis at the junction of the two DNA regions
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
