Ctr6, a Vacuolar Membrane Copper Transporter inSchizosaccharomyces pombe

Abstract

Aerobic organisms possess efficient systems for the transport of copper. This involves transporters that mediate the passage of copper across biological membranes to reach essential intracellular copper-requiring enzymes. In this report, we identify a new copper transporter in Schizosaccharomyces pombe, encoded by the ctr6 ؉ gene. The transcription of ctr6 ؉ is induced under copper-limiting conditions. This regulation is mediated by the cis-acting promoter element CuSE (copper-signaling element) through the copper-sensing transcription factor Cuf1. An S. pombe strain bearing a disrupted ctr6⌬ allele displays a strong reduction of copper,zinc superoxide dismutase activity. When the ctr6؉ gene is overexpressed from the thiamine-inducible nmt1 ؉ promoter, the cells are unable to grow on medium containing exogenous copper. Surprisingly, this copper-sensitive growth phenotype is not due to an increase of copper uptake at the cell surface. Instead, copper delivery across the plasma membrane is reduced. Consistently, this results in repressing ctr4 ؉ gene expression. By using a functional ctr6 ؉ epitope-tagged allele expressed under the control of its own promoter, we localize the Ctr6 protein on the membrane of vacuoles. Furthermore, we demonstrate that Ctr6 is an integral membrane protein that can trimerize. Moreover, we show that Ctr6 harbors a putative copper-binding Met-X-His-Cys-X-Met-X-Met motif in the amino terminus, which is essential for its function. Our findings suggest that under conditions in which copper is scarce, Ctr6 is required as a means to mobilize stored copper from the vacuole to the cytosol.

Deregulated Copper Transport Affects Arabidopsis Development Especially in the Absence of Environmental Cycles

“…Although previous results indicate that the Arabidopsis COPT1 protein could function in Cu acquisition in the plasma membrane of specific plant cells (Kampfenkel et al, 1995;Sancenon et al, 2003Sancenon et al, , 2004, the fact that certain CTR members are located at intracellular endosomal vesicles or vacuolar membranes in other eukaryotic organisms (Bellemare et al, 2002;Rees et al, 2004;van den Berghe et al, 2007;Bertinato et al, 2008) prompted us to address the subcellular localization of COPT1 in Arabidopsis cells. To that end, the coding sequence of the COPT1 protein was fused to GFP (COPT1-GFP) under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter.…”

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

“…In response to copper limitation, Ctr6 appears to mediate the efflux of usable copper from the vacuole into the cytosol (56). Like the ctr4 ϩ and ctr5 ϩ genes, ctr6 ϩ expression is regulated by the Cuf1 transcription factor and is induced in copper-limited cells (54,56,57). Cuf1 plays an essential role in coordinating the copper-dependent transcriptional regulation of copper transporter gene expression in S. pombe.…”

“…Recently, we have identified a gene in fission yeast, ctr6 ϩ , which encodes an intracellular vacuolar copper transporter (56). In response to copper limitation, Ctr6 appears to mediate the efflux of usable copper from the vacuole into the cytosol (56). Like the ctr4 ϩ and ctr5 ϩ genes, ctr6 ϩ expression is regulated by the Cuf1 transcription factor and is induced in copper-limited cells (54,56,57).…”

“…Within this complex, the exact function of each protein is currently unclear. Recently, we have identified a gene in fission yeast, ctr6 ϩ , which encodes an intracellular vacuolar copper transporter (56). In response to copper limitation, Ctr6 appears to mediate the efflux of usable copper from the vacuole into the cytosol (56).…”

Les bases moléculaires de l’approvisionnement en cuivre

“…La présence de motifs Met, dans l'une ou l'autre des protéines, est suffisante pour l'incorporation du cuivre chez S. pombe [6]. Un troisième transporteur nommé Ctr6 est présent dans la membrane vacuolaire [7]. Son action est similaire à celle de la protéine Ctr2 chez S. cerevisiae.…”