Cobbold Christian, Coventry Julie, Ponnambalam Sreenivasan, Monaco Anthony P
Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford 0X3 7BN, UK.
Hum Mol Genet. 2003 Jul 1;12(13):1523-33. doi: 10.1093/hmg/ddg166.
The Menkes disease gene encodes a P-type transmembrane ATPase (ATP7A) that translocates cytosolic copper ions across intracellular membranes of compartments along the secretory pathway. ATP7A moves from the trans-Golgi network (TGN) to the cell surface in response to exogenously added copper ions and recycles back to the TGN upon copper removal. The protein contains a C-terminal di-leucine motif necessary for internalization from the cell surface. In this study we show that ATP7A is internalized by a novel pathway that is independent of clathrin-mediated endocytosis. Expression of dominant-negative mutants of the dynamin-I, dynamin-II and Eps15 proteins that block clathrin-dependent endocytosis of the transferrin receptor do not inhibit internalization of endogenous ATP7A, or an ATP7A reporter molecule (CD8-MCF1). Similarly, inhibitors of caveolae-mediated uptake do not affect ATP7A internalization whilst preventing uptake of PODIPY-ganglioside GM(1), a caveolae marker. In contrast, expression of a constitutively active mutant of the Rac1 GTPase inhibits plasma membrane internalization of both the ATP7A and transferrin receptor transmembrane proteins. These findings define a novel route required for ATP7A internalization and delivery to endosomes.
门克斯病基因编码一种P型跨膜ATP酶（ATP7A），该酶可将胞质铜离子转运穿过分泌途径中各间隔的细胞内膜。ATP7A会响应外源添加的铜离子，从反式高尔基体网络（TGN）转移至细胞表面，并在铜去除后循环回到TGN。该蛋白质含有一个C末端双亮氨酸基序，这是其从细胞表面内化所必需的。在本研究中，我们表明ATP7A通过一种独立于网格蛋白介导的内吞作用的新途径进行内化。抑制转铁蛋白受体的网格蛋白依赖性内吞作用的发动蛋白-I、发动蛋白-II和Eps15蛋白的显性负性突变体的表达，并不抑制内源性ATP7A或ATP7A报告分子（CD8-MCF1）的内化。同样，小窝介导摄取的抑制剂在阻止小窝标记物PODIPY-神经节苷脂GM(1)摄取的同时，并不影响ATP7A的内化。相反，Rac1 GTP酶的组成型活性突变体的表达抑制了ATP7A和转铁蛋白受体跨膜蛋白两者从质膜的内化。这些发现确定了ATP7A内化并转运至内体所需的一条新途径。
