Genomic analysis of severe hypersensitivity to hygromycin B reveals linkage to vacuolar defects and new vacuolar gene functions in Saccharomyces cerevisiae
Genomic analysis of severe hypersensitivity to hygromycin B reveals linkage to vacuolar defects and new vacuolar gene functions in Saccharomyces cerevisiae
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DOI：
10.1007/s00294-009-0285-3
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发表时间：
2010-04-01
期刊：
影响因子：
2.5
通讯作者：
Gharakhanian, Editte
中科院分区：
文献类型：
--
作者：
Banuelos, M. G.;Moreno, D. E.;Gharakhanian, Editte
酿酒酵母的液泡是研究溶酶体运输、生物发生和功能的重要模型。几种在这种空泡事件中有缺陷的酵母突变体已经不能在低水平的潮霉素B（一种氨基糖苷类抗生素）下生长。我们假设，这种严重的超敏潮霉素B（hhy）与空泡缺陷，并进行了基因组筛选的表型使用非必需基因的单倍体缺失菌株库。初步鉴定了14个HHY基因，并进行了生物信息学分析。对发现的hhy突变体进行了实验表征，包括囊泡运输、液泡形态以及在各种应激和药物条件下的生长。生物信息学分析和表型特征的结合暗示了所有hhy突变体中囊泡运输、液泡融合/分裂或液泡功能的缺陷。富集收集物对莫能菌素的敏感性，表明空泡运输缺陷。此外，所有hhy突变体显示出对雷帕霉素和咖啡因的严重敏感性，提示TOR激酶途径缺陷。我们的实验结果还建立了一个新的作用，在空泡和囊泡功能的两个基因：PAF 1，编码RNAP II相关蛋白的细胞周期调控基因的表达所需的，和TPD 3，编码蛋白磷酸酶2A的调节亚基。因此，我们的研究结果支持潮霉素B的严重超敏反应和空泡缺陷之间的联系。
The vacuole of Saccharomyces cerevisiae has been a seminal model for studies of lysosomal trafficking, biogenesis, and function. Several yeast mutants defective in such vacuolar events have been unable to grow at low levels of hygromycin B, an aminoglycoside antibiotic. We hypothesized that such severe hypersensitivity to hygromycin B (hhy) is linked to vacuolar defects and performed a genomic screen for the phenotype using a haploid deletion strain library of non-essential genes. Fourteen HHY genes were initially identified and were subjected to bioinformatics analyses. The uncovered hhy mutants were experimentally characterized with respect to vesicular trafficking, vacuole morphology, and growth under various stress and drug conditions. The combination of bioinformatics analyses and phenotypic characterizations implicate defects in vesicular trafficking, vacuole fusion/fission, or vacuole function in all hhy mutants. The collection was enriched for sensitivity to monensin, indicative of vacuolar trafficking defects. Additionally, all hhy mutants showed severe sensitivities to rapamycin and caffeine, suggestive of TOR kinase pathway defects. Our experimental results also establish a new role in vacuolar and vesicular functions for two genes: PAF1, encoding a RNAP II-associated protein required for expression of cell cycle-regulated genes, and TPD3, encoding the regulatory subunit of protein phosphatase 2A. Thus, our results support linkage between severe hypersensitivity to hygromycin B and vacuolar defects.