The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels
The Caulobacter crescentus GTPase CgtAC is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels
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DOI：
10.1111/j.1365-2958.2004.04354.x
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发表时间：
2004-12-01
影响因子：
3.6
通讯作者：
Maddock, JR
中科院分区：
文献类型：
--
作者：
Datta, K;Skidmore, JM;Maddock, JR
细菌 GTP 结合蛋白的 Obg 亚家族在生化上与 Ras 样蛋白不同，这增加了它们不受常规鸟嘌呤核苷酸交换因子 (GEF) 和/或鸟嘌呤核苷酸激活蛋白 (GAP) 控制的可能性。为了检验这一假设，我们在新月柄杆菌 obg 基因 (cgtA(C)) 中产生了突变，在 Ras 样蛋白中，该突变会导致激活或显性失活表型。在 C. crescentus 中，P168V 突变体在体内不被激活，尽管在体外，P168V 蛋白显示出对 GDP 的亲和力适度降低。 S173N 和 N280Y 突变均未导致显性阴性表型。此外，S173N 的 GTP 结合显着受损，这与该残基在 GTP 结合中的关键作用一致。然而，一般来说，GTP 结合袋中的保守氨基酸对于功能很重要。为了检查消耗 CgtA(C) 的体内后果，我们产生了温度敏感突变体 G80E。在允许的温度下，G80E 细胞生长缓慢，50S 核糖体亚基水平降低，表明 CgtA(C) 对于 50S 组装和/或稳定性很重要。令人惊讶的是，在不允许的温度下，G80E 细胞迅速失去活力，但没有表现出额外的核糖体缺陷。因此，cgtA(C)基因的本质性质似乎并不是由其核糖体功能引起的。 G80E 细胞停滞为分裂前细胞和无柄细胞。同步细胞的流式细胞术显示 G1-S 停滞。因此，CgtA(C) 对于 DNA 复制和细胞周期进展是必需的。
The Obg subfamily of bacterial GTP-binding proteins are biochemically distinct from Ras-like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, we generated mutations in the Caulobacter crescentus obg gene (cgtA(C)) which, in Ras-like proteins, would result in either activating or dominant negative phenotypes. In C. crescentus, a P168V mutant is not activating in vivo, although in vitro, the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Furthermore, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP-binding pocket were, however, important for function. To examine the in vivo consequences of depleting CgtA(C), we generated a temperature-sensitive mutant, G80E. At the permissive temperature, G80E cells grow slowly and have reduced levels of 50S ribosomal subunits, indicating that CgtA(C) is important for 50S assembly and/or stability. Surprisingly, at the non-permissive temperature, G80E cells rapidly lose viability and yet do not display an additional ribosome defect. Thus, the essential nature of the cgtA(C) gene does not appear to result from its ribosome function. G80E cells arrest as predivisional cells and stalkless cells. Flow cytometry on synchronized cells reveals a G1-S arrest. Therefore, CgtA(C) is necessary for DNA replication and progression through the cell cycle.