Peter Chardeen, Laliberté Julie, Beaudoin Jude, Labbé Simon
Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada.
Eukaryot Cell. 2008 Oct;7(10):1781-94. doi: 10.1128/EC.00230-08. Epub 2008 Aug 22.
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.
铜胺氧化酶（CAOs）被认为参与了异源生物胺和生物胺的代谢。其活性对铜的需求是绝对的。在裂殖酵母粟酒裂殖酵母中，cao1(+)和cao2(+)基因被预测编码CAO家族的成员。虽然这两个基因在野生型细胞中都有表达，但我们确定只有cao1(+)的表达会产生一种活性酶，而cao2(+)则不会。定点诱变确定了Cao1 C端区域内的三个组氨酸残基，它们是胺氧化酶活性所必需的。通过使用保留野生型功能的cao1(+)-GFP等位基因，Cao1-GFP定位于细胞质中（GFP是绿色荧光蛋白）。在铜限制条件下，ctr4(+)、ctr5(+)和cuf1(+)的破坏导致胺氧化酶活性出现缺陷，表明功能活跃的Cao1需要Ctr4/5介导的铜转运和转录因子Cuf1。同样，atx1缺失细胞的胺氧化酶活性水平大幅下降。相比之下，ccc2、cox17和pccs的缺失对Cao1活性没有显著影响。通过恢复atx1(+)等位基因，缺乏atx1(+)的细胞中的残余胺氧化酶活性可以恢复到正常水平，这突出了细胞中Atx1存在的至关重要性。使用双杂交分析，我们证明Cao1与Atx1发生物理相互作用，并且这种关联与Atx1与Ccc2 N端区域的关联相当。总的来说，这些结果描述了Atx1作为Ccc2以外分子的铜载体的能力的第一个例子，以及它在将铜传递给Cao1中的关键作用。
