Benghezal M, Lipke P N, Conzelmann A
Institute of Biochemistry, University of Fribourg, Switzerland.
J Cell Biol. 1995 Sep;130(6):1333-44. doi: 10.1083/jcb.130.6.1333.
Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are synthesized by the posttranslational attachment of a preformed glycolipid to newly made glycoproteins. alpha-Agglutinin is a GPI-anchored glycoprotein that gets expressed at the cell surface of MAT alpha cells after induction with type a mating factor. Mutants affecting the biosynthesis of GPI anchors were obtained by selecting for the absence of alpha-agglutinin from the cell wall after induction with a-factor at 37 degrees C. 10 recessive mutants were grouped into 6 complementation classes, gpi4 to gpi9. Mutants are considered to be deficient in the biosynthesis of GPI anchors, since each mutant accumulates an abnormal, incomplete GPI glycolipid containing either zero, two, or four mannoses. One mutant accumulates a complete precursor glycolipid, suggesting that it might be deficient in the transfer of complete precursor lipids to proteins. When labeled with [2-3H]inositol, mutants accumulate reduced amounts of radiolabeled GPI-anchored proteins, and the export of the GPI-anchored Gas1p out of the ER is severely delayed in several mutant strains. On the other hand, invertase and acid phosphatase are secreted by all but one mutant. All mutants show an increased sensitivity to calcofluor white and hygromycin B. This suggests that GPI-anchored proteins are required for the integrity of the yeast cell wall.
糖基磷脂酰肌醇（GPI）锚定膜蛋白是通过将预先形成的糖脂翻译后连接到新合成的糖蛋白上而合成的。α-凝集素是一种GPI锚定糖蛋白，在用a型交配因子诱导后在MATα细胞的细胞表面表达。通过在37℃用a因子诱导后从细胞壁中筛选出不存在α-凝集素，获得了影响GPI锚生物合成的突变体。10个隐性突变体被分为6个互补类，即gpi4至gpi9。突变体被认为在GPI锚的生物合成中存在缺陷，因为每个突变体都积累了一种异常的、不完整的GPI糖脂，其含有零个、两个或四个甘露糖。一个突变体积累了一种完整的前体糖脂，这表明它可能在将完整的前体脂质转移到蛋白质方面存在缺陷。当用[2-³H]肌醇标记时，突变体积累的放射性标记的GPI锚定蛋白量减少，并且在几个突变菌株中，GPI锚定的Gas1p从内质网的输出严重延迟。另一方面，除了一个突变体外，所有突变体都分泌蔗糖酶和酸性磷酸酶。所有突变体对荧光增白剂和潮霉素B的敏感性都增加。这表明GPI锚定蛋白对于酵母细胞壁的完整性是必需的。
