Mechanisms of copper loading on the Schizosaccharomyces pombe copper amine oxidase 1 expressed in Saccharomyces cerevisiae

Abstract

Copper amine oxidases (CAOs) are found in almost every living kingdom. Although Saccharomyces cerevisiae is one of the few yeast species that lacks an endogenous CAO, heterologous gene expression of CAOs from other organisms produces a functional enzyme. To begin to characterize their function and mechanisms of copper acquisition, two putative cao(+) genes from Schizosaccharomyces pombe were expressed in S. cerevisiae. Expression of spao1(+) resulted in the production of an active enzyme capable of catalysing the oxidative deamination of primary amines. On the other hand, expression of spao2(+) failed to produce an active CAO. Using a functional spao1(+)-GFP fusion allele, the SPAO1 protein was localized in the cytosol. Under copper-limiting conditions, yeast cells harbouring deletions of the MAC1, CTR1 and CTR3 genes were defective in amine oxidase activity. Likewise, atx1Delta null cells exhibited no CAO activity, while ccc2Delta mutant cells exhibited decreased levels of amine oxidase activity, and mutations in cox17Delta and ccs1Delta did not cause any defects in this activity. Copper-deprived S. cerevisiae cells expressing spao1(+) required a functional atx1(+) gene for growth on minimal medium containing ethylamine as the sole nitrogen source. Under these conditions, the inability of the atx1Delta cells to utilize ethylamine correlated with the lack of SPAO1 activity, in spite of the efficient expression of the protein. Cells carrying a disrupted ccc2Delta allele exhibited only weak growth on ethylamine medium containing a copper chelator. The results of these studies reveal that expression of the heterologous spao1(+) gene in S. cerevisiae is required for its growth in medium containing ethylamine as the sole nitrogen source, and that expression of an active Schiz. pombe SPAO1 protein in S. cerevisiae depends on the acquisition of copper through the high-affinity copper transporters Ctr1 and Ctr3, and the copper chaperone Atx1.