Rodríguez-Peña J M, Cid V J, Arroyo J, Nombela C
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain.
Mol Cell Biol. 2000 May;20(9):3245-55. doi: 10.1128/MCB.20.9.3245-3255.2000.
The Saccharomyces cerevisiae Ygr189c, Yel040w, and Ylr213c gene products show significant homologies among themselves and with various bacterial beta-glucanases and eukaryotic endotransglycosidases. Deletion of the corresponding genes, either individually or in combination, did not produce a lethal phenotype. However, the removal of YGR189c and YEL040w, but not YLR213c, caused additive sensitivity to compounds that interfere with cell wall construction, such as Congo red and Calcofluor White, and overexpression of YEL040w led to resistance to these compounds. These genes were renamed CRH1 and CRH2, respectively, for Congo red hypersensitive. By site-directed mutagenesis we found that the putative glycosidase domain of CRH1 was critical for its function in complementing hypersensitivity to the inhibitors. The involvement of CRH1 and CRH2 in the development of cell wall architecture was clearly shown, since the alkali-soluble glucan fraction in the crh1Delta crh2Delta strain was almost twice the level in the wild-type. Interestingly, the three genes were subject to different patterns of transcriptional regulation. CRH1 and YLR213c (renamed CRR1, for CRH related) were found to be cell cycle regulated and also expressed under sporulation conditions, whereas CRH2 expression did not vary during the mitotic cycle. Crh1 and Crh2 are localized at the cell surface, particularly in chitin-rich areas. Consistent with the observed expression patterns, Crh1-green fluorescent protein was found at the incipient bud site, around the septum area in later stages of budding, and in ascospore envelopes. Crh2 was found to localize mainly at the bud neck throughout the whole budding cycle, in mating projections and zygotes, but not in ascospores. These data suggest that the members of this family of putative glycosidases might exert a common role in cell wall organization at different stages of the yeast life cycle.
酿酒酵母的Ygr189c、Yel040w和Ylr213c基因产物彼此之间以及与各种细菌β-葡聚糖酶和真核生物内转糖基酶具有显著的同源性。单独或组合缺失相应基因均未产生致死表型。然而，去除YGR189c和YEL040w（而非YLR213c）会导致对干扰细胞壁构建的化合物（如刚果红和荧光增白剂）产生累加敏感性，并且YEL040w的过表达导致对这些化合物产生抗性。这些基因分别被重新命名为CRH1和CRH2，意为对刚果红过敏。通过定点诱变，我们发现CRH1的推定糖苷酶结构域对其在补充对抑制剂的超敏感性方面的功能至关重要。CRH1和CRH2参与细胞壁结构发育这一点得到了明确证明，因为crh1Δcrh2Δ菌株中碱溶性葡聚糖部分几乎是野生型的两倍。有趣的是，这三个基因受到不同模式的转录调控。发现CRH1和YLR213c（重新命名为CRR1，与CRH相关）受细胞周期调控，并且在孢子形成条件下也有表达，而CRH2的表达在有丝分裂周期中没有变化。Crh1和Crh2定位于细胞表面，特别是在富含几丁质的区域。与观察到的表达模式一致，在芽起始位点、出芽后期的隔膜区域周围以及子囊孢子包膜中发现了Crh1-绿色荧光蛋白。发现Crh2在整个出芽周期中主要定位于芽颈、交配突起和合子中，但不在子囊孢子中。这些数据表明，这个推定糖苷酶家族的成员可能在酵母生命周期的不同阶段对细胞壁组织发挥共同作用。
