Spurway T D, Dalley J A, High S, Bulleid N J
University of Manchester, School of Biological Sciences, 2.205 Stopford Building, Manchester M13 9PT, United Kingdom.
J Biol Chem. 2001 May 11;276(19):15975-82. doi: 10.1074/jbc.M010128200. Epub 2001 Feb 23.
The addition of glycosylphosphatidylinositol (GPI) anchors to proteins occurs by a transamidase-catalyzed reaction mechanism soon after completion of polypeptide synthesis and translocation. We show that placental alkaline phosphatase becomes efficiently GPI-anchored when translated in the presence of semipermeabilized K562 cells but is not GPI-anchored in cell lines defective in the transamidase subunit hGpi8p. By studying the synthesis of placental alkaline phosphatase, we demonstrate that folding of the protein is not influenced by the addition of a GPI anchor and conversely that GPI anchor addition does not require protein folding. These results demonstrate that folding of the ectodomain and GPI addition are two distinct processes and can be mutually exclusive. When GPI addition is prevented, either by synthesis of the protein in the presence of cell lines defective in GPI addition or by mutation of the GPI carboxyl-terminal signal sequence cleavage site, the substrate forms a prolonged association with the transamidase subunit hGpi8p. The ability of the transamidase to recognize and associate with GPI anchor signal sequences provides an explanation for the retention of GPI-anchored protein within the ER in the absence of GPI anchor addition.
糖基磷脂酰肌醇（GPI）锚添加到蛋白质上的过程，是在多肽合成和转运完成后不久，通过转酰胺酶催化的反应机制进行的。我们发现，胎盘碱性磷酸酶在半透性K562细胞存在的情况下进行翻译时能有效地进行GPI锚定，但在转酰胺酶亚基hGpi8p有缺陷的细胞系中则不能进行GPI锚定。通过研究胎盘碱性磷酸酶的合成，我们证明蛋白质的折叠不受GPI锚添加的影响，反之，GPI锚的添加也不需要蛋白质折叠。这些结果表明，胞外结构域的折叠和GPI添加是两个不同的过程，并且可能相互排斥。当GPI添加被阻止时，无论是通过在GPI添加有缺陷的细胞系存在的情况下合成蛋白质，还是通过GPI羧基末端信号序列切割位点的突变，底物都会与转酰胺酶亚基hGpi8p形成长时间的结合。转酰胺酶识别并与GPI锚信号序列结合的能力，为在没有GPI锚添加的情况下，GPI锚定蛋白保留在内质网中提供了解释。
