Biosynthesis pathway of GPI anchored proteins.

许多真核细胞表面蛋白通过糖基磷脂酰肌醇(GPI)锚定在质膜上。GPI转氨酶通过用预先组装的GOI取代蛋白质的C-末端GPI连接信号肽，介导GPI锚定在内质网中。已知GPI转氨酶是一个含有GAAI和GPI8的复合酶，为了进一步研究GPI转氨酶的性质，我们对该酶复合体进行了亲和纯化，发现了两个新的组分：PIG-S和PIG-T。为了确定PIG-S和PIG-T的作用，我们通过同源重组的方法在小鼠F9细胞中干扰了这些基因。PIG-S和PIG-T基因敲除细胞在GPI向蛋白质的转移方面存在缺陷。我们建立了中国仓鼠卵巢细胞，代表了GPI锚定蛋白缺失突变体的一个新的互补群U类。U类细胞积累了成熟和未成熟的GPI，在体外不具有GPI转氨酶活性。我们克隆了相关基因，命名为PIG-U，编码一个435个氨基酸的疏水蛋白。从表达表位标记的GPI8的细胞中纯化的GPI转氨酶复合体含有PIG-U和其他四种已知成分。缺乏PIG-U的细胞通常会形成其他四种成分的复合体，但不能切割GPI附着信号肽。因此，这些结果表明GPI转氨酶由5种蛋白质组成。我们还证明了两个亚基，GPI8和GPI-T，通过保守的半胱氨酸残基形成了功能上重要的二硫键。

Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a prolacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GOI. It was known that the GPI transamidase is a complex containing GAAI and GPI8 To further characterize GPI transamidase, we affinity-purified the enzyme complex and found two new components PIG-S and PIG-T. To determinc roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins. We established Chinese hamster ovary cells representing a new complementation group of GPI-anchored protein-deficient mutants, class U. The class U cells accumulated mature and immature GPI and did not have in vitro GPI transamidase activity. We cloned the responsible gene, termed PIG-U, that encodes a 435-amino-acid hydrophobic protein. GPI transamidase complex affnity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U as well as four other known components. Cells lacking PIG-U normally formed complexes of four other components but had no ability to cleave the GPI attachment signal peptide. Therefore, these results demonstrated that GPI transamidase consists of five proteins. We also demonstrated that two subunits, GPI8 and GPI-T, from a functionally important disulfide bond through conserved cysteine residues.