Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity
Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity
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DOI：
10.1128/mcb.13.10.6012-6023.1993
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发表时间：
1993-10
影响因子：
5.3
通讯作者：
Robert Cafferkey;P. Young;M. McLaughlin;D. Bergsma;Y. Koltin;G. Sathe;L. Faucette;W. Eng;R. Johnson;G. P. Livi
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文献类型：
--
作者：
Robert Cafferkey;P. Young;M. McLaughlin;D. Bergsma;Y. Koltin;G. Sathe;L. Faucette;W. Eng;R. Johnson;G. P. Livi
雷帕霉素是一种大环内酯类抗真菌剂，具有有效的免疫抑制特性。在酿酒酵母中，雷帕霉素敏感性由特异性细胞质受体介导，该受体是人FKBP 12（hFKBP 12）的同源物。酵母FKBP 12（RBP 1）基因的缺失导致隐性耐药性，hFKBP 12的表达恢复雷帕霉素敏感性。这些数据支持FKBP 12和雷帕霉素形成毒性复合物破坏其他细胞蛋白质功能的想法。为了鉴定这些蛋白质，我们在野生型单倍体和二倍体细胞以及经工程改造表达hFKBP 12的单倍体rbp 1：：URA 3细胞中分离出显性雷帕霉素抗性突变体。遗传分析表明，显性突变与RBP 1突变是非等位的，并定义了两个基因，命名为DRR 1和DRR 2（显性雷帕霉素抗性）。从基因组YCp 50文库中克隆DRR 1和DRR 2的突变拷贝，通过它们在野生型细胞中赋予耐药性的能力。对突变型drr 1等位基因的DNA序列分析揭示了一个长的开放阅读框架，预测了一种新的2470个氨基酸的蛋白质，该蛋白质具有几个基序，表明参与细胞内信号转导，包括靠近N末端的亮氨酸拉链，两个推定的DNA结合序列，和一个与酵母（VPS 34）和牛磷脂酰肌醇3-激酶的110-kDa催化亚基具有显著序列相似性的结构域。在突变体单倍体菌株中DRR 1的基因组破坏恢复了药物敏感性，并证明该基因编码非必需的功能。7个独立的drr 1dom等位基因的DNA序列比较确定在同一密码子内的磷脂酰肌醇3-激酶结构域中的单碱基对取代，导致Ser-1972的变化为Arg或Asn。我们得出结论，DRR 1（单独或与DRR 2组合）作为FKBP 12-雷帕霉素复合物的靶点，或者DRR 1中的错义突变使其能够补偿正常药物靶点的功能。
Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.