tRNA and protein methylase complexes mediate zymocin toxicity in yeast
tRNA and protein methylase complexes mediate zymocin toxicity in yeast
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DOI：
10.1111/j.1365-2958.2008.06358.x
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发表时间：
2008-09-01
影响因子：
3.6
通讯作者：
Schaffrath, Raffael
中科院分区：
文献类型：
--
作者：
Studte, Patrick;Zink, Sabrina;Schaffrath, Raffael
在摇摆尿苷（U34）位置的酿酒酵母tRNA反密码子的修饰是由来自乳酸克鲁维酵母的发酵菌素tRNase杀伤毒素切割tRNA所必需的。因此，U34修饰缺陷，包括缺乏U34 tRNA甲基转移酶Trm 9，保护免受tRNA切割和酶蛋白。使用zymocin作为工具，我们已经确定了TRM 9中可能影响U34甲基化反应的毒素抗性突变。最引人注目的是，Trm 9中的C-末端截短消除了与Trm 112的相互作用，Trm 112是一种显示出单独用Lys 9和另外两种甲基化酶Trm 11和Mtq 2纯化的蛋白质。GAL 1-TRM 112等位基因的下调可保护细胞不受酵母菌素的影响，而LYS 9、TRM 11和MTQ 2是酵母菌素的剂量抑制剂。基于免疫沉淀研究，后一种情况与Trm 112的竞争相关，并且过量时，这些Trm 112伴侣中的一些干扰毒素相关的Trm9.Trm112复合物的形成。与trm 11 Delta或lys 9 Delta细胞相反，trm 112 Delta和mtq 2 Delta无效突变体是抗发酵素的。与Mtq 2甲基化Sup 45对释放因子二聚体Sup45.Sup35翻译终止的作用一致，我们观察到Sup 45过表达和Sup 45突变体抑制了酶原。有趣的是，这种抑制与由zymocin的tRNase活性靶向的tRNA种类的上调水平相关。
Modification of Saccharomyces cerevisiae tRNA anticodons at the wobble uridine (U34) position is required for tRNA cleavage by the zymocin tRNase killer toxin from Kluyveromyces lactis. Hence, U34 modification defects including lack of the U34 tRNA methyltransferase Trm9 protect against tRNA cleavage and zymocin. Using zymocin as a tool, we have identified toxin-resistant mutations in TRM9 that are likely to affect the U34 methylation reaction. Most strikingly, C-terminal truncations in Trm9 abolish interaction with Trm112, a protein shown to individually purify with Lys9 and two more methylases, Trm11 and Mtq2. Downregulation of a GAL1-TRM112 allele protects against zymocin whereas LYS9, TRM11 and MTQ2 are dosage suppressors of zymocin. Based on immune precipitation studies, the latter scenario correlates with competition for Trm112 and in excess, some of these Trm112 partners interfere with formation of the toxin-relevant Trm9.Trm112 complex. In contrast to trm11 Delta or lys9 Delta cells, trm112 Delta and mtq2 Delta null mutants are zymocin resistant. In line with the identified role that methylation of Sup45 by Mtq2 has for translation termination by the release factor dimer Sup45.Sup35, we observe that SUP45 overexpression and sup45 mutants suppress zymocin. Intriguingly, this suppression correlates with upregulated levels of tRNA species targeted by zymocin's tRNase activity.