Castrejon F, Gomez A, Sanz M, Duran A, Roncero C
Departamento de Microbiologia y Genetica, CSIC/Universidad de Salamanca, Edificio Departamental, R-219, Avda. Campo Charro s/n, 37007-Salamanca, Spain.
Eukaryot Cell. 2006 Mar;5(3):507-17. doi: 10.1128/EC.5.3.507-517.2006.
The Saccharomyces cerevisiae ynl294cDelta (rim21Delta) mutant was identified in our lab owing to its moderate resistance to calcofluor, although it also displayed all of the phenotypic traits associated with its function as the putative sensor (Rim21p) of the RIM101 pathway. rim21Delta also showed moderate hypersensitivity to sodium dodecyl sulfate, caffeine, and zymolyase, and the cell wall compensatory response in this mutant was very poor, as indicated by the almost complete absence of Slt2 phosphorylation and the modest increase in chitin synthesis after calcofluor treatment. However, the cell integrity pathway appeared functional after caffeine treatment or thermal stress. rim21Delta and rim101Delta mutant strains shared all of the cell-wall-associated phenotypes, which were reverted by the expression of Rim101-531p, the constitutively active form of this transcription factor. Therefore, the absence of a functional RIM101 pathway leads to cell wall defects. rim21Delta, as well as rim101Delta, was synthetic lethal with slt2Delta, a synthetic defect alleviated by osmotic stabilization of the media. The double mutants grown in osmotically stabilized media were extremely hypersensitive to zymolyase and showed thicker cell walls, with poorly defined mannoprotein layers. In contrast, rim21Delta rlm1Delta and rim101Delta rlm1Delta double mutants were fully viable. Taken together, these results show that the RIM101 pathway participates directly in cell wall assembly and that it acts in parallel with the protein kinase C pathway (PKC) in this process independently of the transcriptional effect of the compensatory response mediated by this route. In addition, these results provide new experimental evidence of the direct involvement of the PKC signal transduction pathway through the Sltp2 kinase in the construction of yeast cell walls.
在我们实验室中鉴定出酿酒酵母ynl294cΔ（rim21Δ）突变体，因其对荧光增白剂具有中等抗性，尽管它也表现出与作为RIM101途径假定传感器（Rim21p）功能相关的所有表型特征。rim21Δ对十二烷基硫酸钠、咖啡因和溶菌酶也表现出中等程度的超敏反应，并且该突变体中的细胞壁补偿反应非常差，荧光增白剂处理后Slt2磷酸化几乎完全缺失以及几丁质合成适度增加表明了这一点。然而，咖啡因处理或热应激后细胞完整性途径似乎是有功能的。rim21Δ和rim101Δ突变菌株具有所有与细胞壁相关的表型，这些表型可通过该转录因子的组成型活性形式Rim101-531p的表达得以恢复。因此，功能性RIM101途径的缺失会导致细胞壁缺陷。rim21Δ以及rim101Δ与slt2Δ是合成致死的，培养基的渗透稳定可缓解这种合成缺陷。在渗透稳定培养基中生长时，双突变体对溶菌酶极度超敏，并且细胞壁更厚，甘露糖蛋白层界定不清。相反，rim21Δrlm1Δ和rim101Δrlm1Δ双突变体完全可存活。综上所述，这些结果表明RIM101途径直接参与细胞壁组装，并且在这个过程中它与蛋白激酶C途径（PKC）平行发挥作用，独立于该途径介导的补偿反应的转录效应。此外，这些结果为PKC信号转导途径通过Sltp2激酶直接参与酵母细胞壁构建提供了新的实验证据。
