Klassen Roland, Paluszynski John P, Wemhoff Sabrina, Pfeiffer Annika, Fricke Julia, Meinhardt Friedhelm
Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstr. 3, D-48149 Münster, Germany.
Mol Microbiol. 2008 Aug;69(3):681-97. doi: 10.1111/j.1365-2958.2008.06319.x. Epub 2008 Jun 28.
The Pichia acaciae killer toxin (PaT) arrests yeast cells in the S-phase of the cell cycle and induces DNA double-strand breaks (DSBs). Surprisingly, loss of the tRNA-methyltransferase Trm9 - along with the Elongator complex involved in synthesis of 5-methoxy-carbonyl-methyl (mcm(5)) modification in certain tRNAs - conferred resistance against PaT. Overexpression of mcm(5)-modified tRNAs identified tRNA(Gln)((UUG)) as the intracellular target. Consistently, toxin-challenged cells displayed reduced levels of tRNA(Gln) and in vitro the heterologously expressed active toxin subunit disrupts the integrity of tRNA(Gln)((UUG)). Other than Kluyveromyces lactis zymocin, an endonuclease specific for tRNA(Glu)((UUC)), affecting its target in a mcm(5)-dependent manner, PaT exerts activity also on tRNA(Gln) lacking such modification. As sensitivity is restored in trm9 elp3 double mutants, target tRNA cleavage is selectively inhibited by incomplete wobble uridine modification, as seen in trm9, but not in elp3 or trm9 elp3 cells. In addition to tRNA(Gln)((UUG)), tRNA(Gln)((CUG)) is also cleaved in vitro and overexpression of the corresponding gene increased resistance. Consistent with tRNA(Gln)((CUG)) as an additional TRM9-independent target, overexpression of PaT's tRNase subunit abolishes trm9 resistance. Most interestingly, a functional DSB repair pathway confers PaT but also zymocin resistance, suggesting DNA damage to occur generally concomitant with specific tRNA offence.
金合欢毕赤酵母杀伤毒素（PaT）使酵母细胞停滞在细胞周期的S期并诱导DNA双链断裂（DSB）。令人惊讶的是，tRNA甲基转移酶Trm9的缺失——连同参与某些tRNA中5-甲氧基羰基甲基（mcm⁵）修饰合成的延伸因子复合物——赋予了对PaT的抗性。mcm⁵修饰的tRNA的过表达确定tRNA⁺Gln⁺（⁺UUG⁺）为细胞内靶点。一致地，受到毒素攻击的细胞显示tRNA⁺Gln⁺水平降低，并且在体外，异源表达的活性毒素亚基破坏了tRNA⁺Gln⁺（⁺UUG⁺）的完整性。除了乳酸克鲁维酵母酶菌素（一种对tRNA⁺Glu⁺（⁺UUC⁺）具有特异性的核酸内切酶，以mcm⁵依赖的方式影响其靶点）之外，PaT也对缺乏这种修饰的tRNA⁺Gln⁺发挥作用。由于在trm9 elp3双突变体中恢复了敏感性，如在trm9中所见，但在elp3或trm9 elp3细胞中未见，不完全的摆动尿苷修饰选择性地抑制了靶tRNA的切割。除了tRNA⁺Gln⁺（⁺UUG⁺）之外，tRNA⁺Gln⁺（⁺CUG⁺）在体外也被切割，并且相应基因的过表达增加了抗性。与tRNA⁺Gln⁺（⁺CUG⁺）作为另一个不依赖TRM9的靶点一致，PaT的tRNase亚基的过表达消除了trm9抗性。最有趣的是，一个功能性的DSB修复途径赋予了对PaT以及酶菌素的抗性，这表明DNA损伤通常与特定的tRNA损伤同时发生。
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