ABCC6 mediates release of ATP from hepatocytes into the blood. Extracellularly, ATP is converted into the mineralization inhibitor pyrophosphate. Consequently, inactivating ABCC6 mutations give low plasma pyrophosphate underlying the ectopic mineralization disorder pseudoxanthoma elasticum. How ABCC6 mediates cellular ATP release is unknown. Mechanistic studies are hampered because fluorophores attached to ABCC6's N- or C-terminus result in intracellular retention and degradation. Here we describe that intramolecular introduction of fluorophores yields fully functional ABCC6 fusion proteins. An ABCC6 variant with the catalytic glutamate of the second nucleotide-binding domain mutated, correctly routed to the plasma membrane, but was inactive. Finally, N-terminal His10 or FLAG tags did not affect the activity of fusion proteins, allowing their purification for biochemical characterization. Hence, these fusion proteins provide excellent tools to study ABCC6 biology.

中文翻译：

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生成功能齐全的荧光融合蛋白以深入了解 ABCC6 生物学

ABCC6 介导 ATP 从肝细胞释放到血液中。在细胞外，ATP 转化为矿化抑制剂焦磷酸盐。因此，失活的 ABCC6 突变在异位矿化障碍弹性假黄瘤的基础上产生低血浆焦磷酸盐。ABCC6 如何介导细胞 ATP 释放尚不清楚。机制研究受到阻碍，因为连接到 ABCC6 的 N 或 C 端的荧光团会导致细胞内滞留和降解。在这里，我们描述了荧光团的分子内引入产生了功能齐全的 ABCC6 融合蛋白。具有第二个核苷酸结合域的催化谷氨酸的 ABCC6 变体发生突变，正确地传递到质膜，但没有活性。最后，N端His10或FLAG标签不影响融合蛋白的活性，允许对其进行纯化以进行生化表征。因此，这些融合蛋白为研究 ABCC6 生物学提供了极好的工具。