van der Vaart J M, Caro L H, Chapman J W, Klis F M, Verrips C T
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
J Bacteriol. 1995 Jun;177(11):3104-10. doi: 10.1128/jb.177.11.3104-3110.1995.
Three glucanase-extractable cell wall proteins from Saccharomyces cerevisiae were purified, and their N-terminal amino acid sequences were determined. With this information, we were able to assign gene products to three known open reading frames (ORFs). The N-terminal sequence of a 55-kDa mannoprotein corresponded with the product of ORF YKL096w, which we named CWP1 (cell wall protein 1). A 80-kDa mannoprotein was identified as the product of the TIP1 gene, and a 180-kDa mannoprotein corresponded to the product of the ORF YKL444, which we named CWP2. CWP1, TIP1, and CWP2 encode proteins of 239, 210, and 92 amino acids, respectively. The C-terminal regions of these proteins all consist for more than 40% of serine/threonine and contain putative glycosylphosphatidylinositol attachment signals. Furthermore, Cwp1p and Tip1p were shown to carry a beta 1,6-glucose-containing side chain. The cwp2 deletion mutant displayed an increased sensitivity to Congo red, calcofluor white, and Zymolyase. Electron microscopic analysis of the cwp2 deletion mutant showed a strongly reduced electron-dense layer on the outside of the cell wall. These results indicate that Cwp2p is a major constituent of the cell wall and plays an important role in stabilizing the cell wall. Depletion of Cwp1p or Tip1p also caused increased sensitivities to Congo red and calcofluor white, but the effects were less pronounced than for cwp2 delta. All three cell wall proteins show a substantial homology with Srp1p, which also appears to be localized in the cell wall. We conclude that these four proteins are small structurally related cell wall proteins.
从酿酒酵母中纯化出三种可被葡聚糖酶提取的细胞壁蛋白，并测定了它们的N端氨基酸序列。根据这些信息，我们能够将基因产物与三个已知的开放阅读框（ORF）对应起来。一种55 kDa甘露糖蛋白的N端序列与ORF YKL096w的产物相对应，我们将其命名为CWP1（细胞壁蛋白1）。一种80 kDa甘露糖蛋白被鉴定为TIP1基因的产物，而一种180 kDa甘露糖蛋白对应于ORF YKL444的产物，我们将其命名为CWP2。CWP1、TIP1和CWP2分别编码239、210和92个氨基酸的蛋白质。这些蛋白质的C端区域都有超过40%的丝氨酸/苏氨酸，并且含有假定的糖基磷脂酰肌醇附着信号。此外，Cwp1p和Tip1p被证明带有含β1,6-葡萄糖的侧链。cwp2缺失突变体对刚果红、荧光增白剂和溶壁酶的敏感性增加。对cwp2缺失突变体的电子显微镜分析表明，细胞壁外侧的电子致密层明显减少。这些结果表明，Cwp2p是细胞壁的主要成分，在稳定细胞壁方面发挥着重要作用。Cwp1p或Tip1p的缺失也导致对刚果红和荧光增白剂的敏感性增加，但影响不如cwp2缺失突变体明显。所有这三种细胞壁蛋白都与Srp1p有很大的同源性，Srp1p似乎也定位在细胞壁中。我们得出结论，这四种蛋白质是结构相关的小细胞壁蛋白。
