Copper Distributed by Atx1 Is Available to Copper Amine Oxidase 1 in Schizosaccharomyces pombe

Abstract

Copper amine oxidases (CAOs) have been proposed to be involved in the metabolism of xenobiotic and biogenic amines. The requirement for copper is absolute for their activity. In the fission yeast Schizosaccharomyces pombe, cao1؉ and cao2 ؉ genes are predicted to encode members of the CAO family. While both genes are expressed in wild-type cells, we determined that the expression of only cao1 ؉ but not cao2 ؉ results in the production of an active enzyme. Site-directed mutagenesis identified three histidine residues within the C-terminal region of Cao1 that are necessary for amine oxidase activity. By use of a cao1 ؉ -GFP allele that retained wild-type function, Cao1-GFP was localized in the cytosol (GFP is green fluorescent protein). Under copper-limiting conditions, disruption of ctr4 ؉ , ctr5 ؉ , and cuf1 ؉ produced a defect in amine oxidase activity, indicating that a functionally active Cao1 requires Ctr4/5-mediated copper transport and the transcription factor Cuf1. Likewise, atx1 null cells exhibited substantially decreased levels of amine oxidase activity. In contrast, deletion of ccc2, cox17, and pccs had no significant effect on Cao1 activity. Residual amine oxidase activity in cells lacking atx1 ؉ can be restored to normal levels by returning an atx1 ؉ allele, underscoring the critical importance of the presence of Atx1 in cells. Using two-hybrid analysis, we demonstrated that Cao1 physically interacts with Atx1 and that this association is comparable to that of Atx1 with the N-terminal region of Ccc2. Collectively, these results describe the first example of the ability of Atx1 to act as a copper carrier for a molecule other than Ccc2 and its critical role in delivering copper to Cao1.

Mfc1 Is a Novel Forespore Membrane Copper Transporter in Meiotic and Sporulating Cells

“…Previous studies have shown that S. pombe possesses two copper-dependent amine oxidases, denoted Cao1 and Cao2 (25). It has been reported that Cao1 requires copper for its activity when expressed in mitotically growing cells (25).…”

“…It has been reported that Cao1 requires copper for its activity when expressed in mitotically growing cells (25). Because Cao1 molecular and cellular characterization has not yet been ascertained in meiosis, we have examined the subcellular location of an active Cao1-GFP fusion protein into an S. pombe h Ϫ /h ϩ cao1⌬/⌬ mutant strain that was induced to undergo azygotic meiosis.…”

“…A combination of observations in S. pombe and Saccharomyces cerevisiae has revealed that Atx1 delivers copper to Ccc2, a P-type ATPase on the Golgi membrane that transports copper into the secretory pathway where it can be incorporated into newly synthesized cuproproteins (24). Using mutant strains and protein-protein interaction assays, our group has reported that the fully active copper amine oxidase Cao1 is dependent on the presence of Atx1 (25). However, the mechanism by which copper is loaded into Cao1 via Atx1 is currently unknown.…”

“…Cell lysates were prepared from each culture, and equal amounts of cellular protein were subjected to a colorimetric assay as described previously (25). In this quantitative assay, hydrogen peroxide that was released from a reaction catalyzed by CAO was determined by a method using 4-aminoantipyrine and vanillic acid to generate a quinone-imine dye, which is detectable at 498 nm.…”

Simon Labbé’s work on iron and copper homeostasis

“…Dr. Labbé's team has demonstrated that the metallochaperone Atx1 directly activates the copper amine oxidase Cao1 [19] . Although it has been shown that Atx1 specifically delivers copper to the copper ATPase Ccc2 in the secretory pathway [20,21] , Dr. Labbé's studies have described the first example of the ability of Atx1 to act as a copper carrier for a molecule other than Ccc2 [22] .…”