The Wilson disease protein, ATP7B maintains copper (herein referring to the Cu⁺ ion) homeostasis in the liver. ATP7B traffics from trans-Golgi network to endolysosomes to export excess copper. Regulation of ATP7B trafficking to and from endolysosomes is not well understood. We investigated the fate of ATP7B after copper export. At high copper levels, ATP7B traffics primarily to acidic, active hydrolase (cathepsin-B)-positive endolysosomes and, upon subsequent copper chelation, returns to the trans-Golgi network (TGN). At high copper, ATP7B colocalizes with endolysosomal markers and with a core member of retromer complex, VPS35. Knocking down VPS35 did not abrogate the copper export function of ATP7B or its copper-responsive anterograde trafficking to vesicles; rather upon subsequent copper chelation, ATP7B failed to relocalize to the TGN, which was rescued by overexpressing wild-type VPS35. Overexpressing mutants of the retromer complex-associated proteins Rab7A and COMMD1 yielded a similar non-recycling phenotype of ATP7B. At high copper, VPS35 and ATP7B are juxtaposed on the same endolysosome and form a large complex that is stabilized by in vivo photoamino acid labeling and UV-crosslinking. We demonstrate that retromer regulates endolysosome to TGN trafficking of copper transporter ATP7B in a manner that is dependent upon intracellular copper.

威尔逊病蛋白 ATP7B 维持肝脏中铜（本文指 Cu ^{+离子）的稳态。}ATP7B 从跨高尔基体网络运输至内溶酶体，以输出多余的铜。ATP7B 进出内溶酶体的运输调控尚不清楚。我们调查了铜出口后 ATP7B 的命运。在高铜水平下，ATP7B 主要运输至酸性、活性水解酶（组织蛋白酶-B）阳性内溶酶体，并在随后的铜螯合后返回至反式高尔基体网络 (TGN)。在高铜含量下，ATP7B 与内溶酶体标记物以及逆转录体复合物的核心成员 VPS35 共定位。敲低 VPS35 并不会消除 ATP7B 的铜输出功能或其铜响应性顺行运输至囊泡；相反，在随后的铜螯合后，ATP7B 未能重新定位到 TGN，而 TGN 是通过过度表达野生型 VPS35 来挽救的。逆转录酶复合体相关蛋白 Rab7A 和 COMMD1 的过表达突变体产生了与 ATP7B 类似的非循环表型。在高铜含量下，VPS35 和 ATP7B 并置在同一内溶酶体上，并形成一个大的复合物，该复合物通过体内光氨基酸标记和紫外线交联而稳定。我们证明，retromer 以依赖于细胞内铜的方式调节铜转运蛋白 ATP7B 的内溶酶体至 TGN 的运输。