Sulfur transfer and activation by ubiquitin-like modifier system Uba4•Urm1 link protein urmylation and tRNA thiolation in yeast.
Sulfur transfer and activation by ubiquitin-like modifier system Uba4•Urm1 link protein urmylation and tRNA thiolation in yeast.
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DOI：
10.15698/mic2016.11.539
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发表时间：
2016-10-24
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影响因子：
--
通讯作者：
Schaffrath R
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作者：
Jüdes A;Bruch A;Klassen R;Helm M;Schaffrath R
Urm1是泛素蛋白家族中一个独特的双功能成员，从酵母到人类都是保守的。它既作为泛素样氨基化的蛋白质修饰剂，又作为tRNA硫基化的硫供体，与延长子途径一起在反密码子中形成5-甲氧基-羰基-甲基-2-硫（mcm5s2）修饰的摆动尿苷（U34）。我们以酿酒酵母为模型研究了这两种功能之间的关系，研究了培养温度和硫供应是否也有助于URM1途径的tRNA硫代化分支。通过对Urm1偶联的监测，我们发现在升高的培养温度或缺硫条件下，过氧化物还蛋白Ahp1的氨基化受到抑制。与此相一致，硫转移缺陷的突变体被发现与硫代羧化（Urm1- cosh）激活Urm1所需的酶（Tum1, Uba4）相关，其Ahp1 urmy化和mcm5s2U34修饰水平急剧降低。此外，通过位点特异性诱变发现，对Urm1-COSH形成至关重要的e1样激活物（Uba4）中的s -转移罗丹斯结构域（RHD）对蛋白质的氨基化和tRNA硫代化至关重要，但不是必需的。总而言之，硫的供应、转移和激活在化学上连接着蛋白质的甲基化和tRNA的硫代化。这些特征将泛素样修饰系统Uba4•Urm1与典型的泛素家族成员区分开来，并将有助于阐明除了它们的机制联系外，Urm1途径的蛋白质和tRNA修饰分支是否也可能在功能上相互关联。
Urm1 is a unique dual-function member of the ubiquitin protein family and conserved from yeast to man. It acts both as a protein modifier in ubiquitin-like urmylation and as a sulfur donor for tRNA thiolation, which in concert with the Elongator pathway forms 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modified wobble uridines (U34) in anticodons. Using Saccharomyces cerevisiae as a model to study a relationship between these two functions, we examined whether cultivation temperature and sulfur supply previously implicated in the tRNA thiolation branch of the URM1 pathway also contribute to proper urmylation. Monitoring Urm1 conjugation, we found urmylation of the peroxiredoxin Ahp1 is suppressed either at elevated cultivation temperatures or under sulfur starvation. In line with this, mutants with sulfur transfer defects that are linked to enzymes (Tum1, Uba4) required for Urm1 activation by thiocarboxylation (Urm1-COSH) were found to maintain drastically reduced levels of Ahp1 urmylation and mcm5s2U34 modification. Moreover, as revealed by site specific mutagenesis, the S-transfer rhodanese domain (RHD) in the E1-like activator (Uba4) crucial for Urm1-COSH formation is critical but not essential for protein urmylation and tRNA thiolation. In sum, sulfur supply, transfer and activation chemically link protein urmylation and tRNA thiolation. These are features that distinguish the ubiquitin-like modifier system Uba4•Urm1 from canonical ubiquitin family members and will help elucidate whether, in addition to their mechanistic links, the protein and tRNA modification branches of the URM1 pathway may also relate in function to one another.