The Schizosaccharomyces pombe Cuf1 Is Composed of Functional Modules from Two Distinct Classes of Copper Metalloregulatory Transcription Factors

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.

The copper-dependent ACE1 transcription factor activates the transcription of the mco1 gene from the basidiomycete Phanerochaete chrysosporium

“…Although some displacement of the complex was observed with the mutant probe, the complex was completely displaced with a 50-fold molar excess of the unlabelled probe possessing the wild-type ACE element. This result indicates that the complex was formed by sequence-specific interactions, as has also been demonstrated for Cuf1, a Schizosaccharomyces pombe transcription factor that has a DNA sequence binding specificity similar to the ACE1 protein from Saccharomyces cerevisiae (Beaudoin et al, 2003). The requirement for Cu + in the incubation mixture is also indicative of the specificity of this binding (Fig.…”

“…All ACE elements adhere to the consensus sequence H(T)HNNGCTGD (H, not G, N, any nucleotide, D, not C). Residues shown in bold type have been assigned a key role involved in DNA-protein interaction (Gralla et al, 1991;Beaudoin et al, 2003). (C) The mco1 promoter region, as defined in Methods.…”

Identification and functional characterisation of ctr1, a Pleurotus ostreatus gene coding for a copper transporter

“…Furthermore, the Cu-signalling element (CuSE) with the consensus sequence 5¢-DWDDHGCTGD-3¢ (D=A, G, or T; H=A, C, or T, and W=T or A) defined in Schizosaccharomyces pombe as a cis-acting element found in the promoter of genes regulated by copper, occurs at position À233 in the promoter of P. ostreatus ctr1. In S. pombe, CuSE is the binding site for the transcription factor encoded by cuf1 (Beaudoin et al 2003). Although the homologous cuf1 gene has not been functionally identified in P. ostreatus, a sequence with significant homology to S. pombe cuf1 has been detected (our unpublished results).…”

The Schizosaccharomyces pombe Pccs Protein Functions in Both Copper Trafficking and Metal Detoxification Pathways

“…Binding studies reveal that the Cuf1 N-terminal 174 amino acids are important for binding to the CuSE (58). A motif containing five clustered cysteine residues near its C terminus constitutes the minimal copper-sensing module of Cuf1 and serves to inactivate Cuf1 function when cells are grown under copper-replete conditions (58).…”