Suzuki Tomoyuki, Ishihara Kaori, Migaki Hitoshi, Matsuura Wataru, Kohda Atsushi, Okumura Katsuzumi, Nagao Masaya, Yamaguchi-Iwai Yuko, Kambe Taiho
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
J Biol Chem. 2005 Jan 7;280(1):637-43. doi: 10.1074/jbc.M411247200. Epub 2004 Nov 2.
Numerous proteins are properly folded by binding with zinc during their itinerary in the biosynthetic-secretory pathway. Several transporters have been implicated in the zinc entry into secretory compartments from cytosol, but their precise roles are poorly understood. We report here that two zinc transporters (ZnT5 and ZnT7) localized in the secretory apparatus are responsible for loading zinc to alkaline phosphatases (ALPs) that are glycosylphosphatidylinositol-anchored membrane proteins exposed to the extracellular site. Disruption of the ZnT5 gene in DT40 cells decreased the ALP activity to 45% of that in the wild-type cells. Disruption of the ZnT7 gene lowered the ALP activity only by 20%. Disruption of both genes markedly decreased the ALP activity to <5%. Overexpression of human ZnT5 or ZnT7 in DT40 cells deficient in both ZnT5 and ZnT7 genes recovered the ALP activity to the level comparable to that in the wild-type cells. The inactive ALP protein in DT40 cells deficient in both ZnT5 and ZnT7 genes was transported to cytoplasmic membrane like the active ALP protein in the wild-type cells. Thus both ZnT5 and ZnT7 contribute to the conversion of apo-ALP to holo-ALP.
许多蛋白质在生物合成-分泌途径中通过与锌结合而正确折叠。几种转运蛋白参与了锌从细胞质溶胶进入分泌区室的过程，但其确切作用尚不清楚。我们在此报告，定位于分泌装置的两种锌转运蛋白（ZnT5和ZnT7）负责将锌加载到碱性磷酸酶（ALP）上，这些碱性磷酸酶是暴露于细胞外位点的糖基磷脂酰肌醇锚定膜蛋白。DT40细胞中ZnT5基因的破坏使ALP活性降至野生型细胞的45%。ZnT7基因的破坏仅使ALP活性降低20%。两个基因的破坏均显著降低ALP活性至<5%。在同时缺乏ZnT5和ZnT7基因的DT40细胞中过表达人ZnT5或ZnT7可使ALP活性恢复到与野生型细胞相当的水平。同时缺乏ZnT5和ZnT7基因的DT40细胞中的无活性ALP蛋白像野生型细胞中的活性ALP蛋白一样被转运到细胞质膜。因此，ZnT5和ZnT7都有助于脱辅基ALP向全酶ALP的转化。
