Bourdineaud J P, van der Vaart J M, Donzeau M, de Sampaïo G, Verrips C T, Lauquin G J
Laboratoire de Physiologie Moléculaire et Cellulaire, IBGC, Centre National de la Recherche Scientifique, Bordeaux, France.
Mol Microbiol. 1998 Jan;27(1):85-98. doi: 10.1046/j.1365-2958.1998.00660.x.
We constructed hybrid proteins containing a plant alpha-galactosidase fused to various C-terminal moieties of the hypoxic Srp1p; this allowed us to identify a cell wall-bound form of Srp1p. We showed that the last 30 amino acids of Srp1p, but not the last 16, contain sufficient information to signal glycosyl-phosphatidylinositol anchor attachment and subsequent cell wall anchorage. The cell wall-bound form was shown to be linked by means of a beta1,6-glucose-containing side-chain. Pmt1p enzyme is known as a protein-O-mannosyltransferase that initiates the O-glycosidic chains on proteins. We found that a pmt1 deletion mutant was highly sensitive to zymolyase and that in this strain the alpha-galactosidase-Srp1 fusion proteins, an alpha-galactosidase-Sed1 hybrid protein and an alpha-galactosidase-alpha-agglutinin hybrid protein were absent from both the membrane and the cell wall fractions. However, the plasma membrane protein Gas1p still receives its glycosyl-phosphatidylinositol anchor in pmt1 cells, and in this mutant strain an alpha-galactosidase-Cwp2 fusion protein was found linked to the cell wall but devoid of beta1,6-glucan side-chain, indicating an alternative mechanism of cell wall anchorage.
我们构建了包含与缺氧Srp1p的各种C末端部分融合的植物α-半乳糖苷酶的杂合蛋白；这使我们能够鉴定出一种细胞壁结合形式的Srp1p。我们发现，Srp1p的最后30个氨基酸而非最后16个氨基酸，包含足以指示糖基磷脂酰肌醇锚定附着及随后细胞壁锚定的信息。结果表明，细胞壁结合形式是通过含β1,6-葡萄糖的侧链连接的。Pmt1p酶是一种已知的蛋白质O-甘露糖基转移酶，可启动蛋白质上的O-糖苷链。我们发现pmt1缺失突变体对溶壁酶高度敏感，并且在该菌株中，膜和细胞壁组分中均不存在α-半乳糖苷酶-Srp1融合蛋白、α-半乳糖苷酶-Sed1杂合蛋白和α-半乳糖苷酶-α-凝集素杂合蛋白。然而，质膜蛋白Gas1p在pmt1细胞中仍能接受其糖基磷脂酰肌醇锚定，并且在该突变菌株中发现一种α-半乳糖苷酶-Cwp2融合蛋白与细胞壁相连，但缺乏β1,6-葡聚糖侧链，这表明存在细胞壁锚定的另一种机制。
