Strausak D, La Fontaine S, Hill J, Firth S D, Lockhart P J, Mercer J F
The Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Parkville, Victoria 3052, Australia.
J Biol Chem. 1999 Apr 16;274(16):11170-7. doi: 10.1074/jbc.274.16.11170.
The Menkes protein (MNK or ATP7A) is a transmembrane, copper-transporting CPX-type ATPase, a subgroup of the extensive family of P-type ATPases. A striking feature of the protein is the presence of six metal binding sites (MBSs) in the N-terminal region with the highly conserved consensus sequence GMXCXXC. MNK is normally located in the trans-Golgi network (TGN) but has been shown to relocalize to the plasma membrane when cells are cultured in media containing high concentrations of copper. The experiments described in this report test the hypothesis that the six MBSs are required for this copper-induced trafficking of MNK. Site-directed mutagenesis was used to convert both cysteine residues in the conserved MBS motifs to serines. Mutation of MBS 1, MBS 6, and MBSs 1-3 resulted in a molecule that appeared to relocalize normally with copper, but when MBSs 4-6 or MBSs 1-6 were mutated, MNK remained in the TGN, even when cells were exposed to 300 microM copper. Furthermore, the ability of the MNK variants to relocalize corresponded well with their ability to confer copper resistance. To further define the critical motifs, MBS 5 and MBS 6 were mutated, and these changes abolished the response to copper. The region from amino acid 8 to amino acid 485 was deleted, resulting in mutant MNK that lacked 478 amino acids from the N-terminal region, including the first four MBSs. This truncated molecule responded normally to copper. Moreover, when either one of the remaining MBS 5 and MBS 6 was mutated to GMXSXXS, the resulting proteins were localized to the TGN in low copper and relocalized in response to elevated copper. These experiments demonstrated that the deleted N-terminal region from amino acid 8 to amino acid 485 was not essential for copper-induced trafficking and that one MBS close to the membrane channel of MNK was necessary and sufficient for the copper-induced redistribution.
门克斯蛋白（MNK或ATP7A）是一种跨膜的、运输铜的CPX型ATP酶，属于庞大的P型ATP酶家族的一个亚组。该蛋白的一个显著特征是在N端区域存在六个金属结合位点（MBS），具有高度保守的共有序列GMXCXXC。MNK通常位于反式高尔基体网络（TGN）中，但已证明当细胞在含有高浓度铜的培养基中培养时，它会重新定位到质膜。本报告中描述的实验检验了以下假设：这六个MBS是铜诱导的MNK转运所必需的。使用定点诱变将保守的MBS基序中的两个半胱氨酸残基转换为丝氨酸。MBS 1、MBS 6和MBSs 1-3的突变产生了一种似乎能正常随铜重新定位的分子，但当MBSs 4-6或MBSs 1-6发生突变时，即使细胞暴露于300微摩尔铜，MNK仍保留在TGN中。此外，MNK变体重新定位的能力与其赋予铜抗性的能力密切相关。为了进一步确定关键基序，对MBS 5和MBS 6进行了突变，这些变化消除了对铜的反应。删除了从氨基酸8到氨基酸485的区域，产生了突变的MNK，其N端区域缺少478个氨基酸，包括前四个MBS。这种截短的分子对铜的反应正常。此外，当剩余的MBS 5和MBS 6中的任何一个突变为GMXSXXS时，产生的蛋白质在低铜条件下定位在TGN中，并在铜浓度升高时重新定位。这些实验表明，从氨基酸8到氨基酸485的缺失N端区域对于铜诱导的转运不是必需的，并且靠近MNK膜通道的一个MBS对于铜诱导的重新分布是必要且充分的。
