Mechanism and significance of ubiquitin-like protein urmylation in yeast

来自出芽酵母的泛素相关修饰剂1 （Urm1）是泛素家族（UbF）的成员，具有真核UbF修饰剂和原核硫载体的蛋白质和tRNA修饰功能。尽管其在tRNA修饰/硫代化中的作用得到了充分的证明，但迄今为止只有一种蛋白（Ahp1）与Urm1结合，强调urmy化的重要性和Urm1的靶标身份在酿酒酵母中都是开放的问题。尽管如此，基于最近对各种人类URM1靶点的分离，将生物学功能分配给蛋白质urmy化似乎是可行的。因此，我们提出了两种互补的策略来研究酵母中的Urm1通路。首先，生物信息学将鉴定人类目标的选定酵母同源物，用于直接酶解和交叉互补分析。这有可能确定真正的Urm1靶点，并研究真核生物中Urm1修饰途径的保守性。第二种方法使用tap标记的Urm1诱饵，通过质谱法在酵母蛋白组范围内鉴定Urm1靶点。为了验证urmyination，将通过独立的Urm1偶联试验评估每种方法的候选物，并且真正的Urm1相互作用与从适当Urm1靶基因突变的菌株中获得的表型谱相关。后者有望探究Urm1修饰途径的相关分支或独立分支，以及Urm1修饰途径中蛋白质urmy化和tRNA硫代化是如何影响靶蛋白功能的。综上所述，通过结合分子、生化和生物信息学方法，该提议可能为真核Urm1修饰途径的生物学和意义提供新的见解。

Ubiquitin-related modifier 1 (Urm1) from budding yeast is a ubiquitin family (UbF) member with protein and tRNA modification functions typical of eukaryal UbF modifiers and prokaryal sulphur carriers, respectively. Despite its well documented role in tRNA modification/thiolation, only one protein (Ahp1) conjugated to Urm1 is known to date from yeast stressing that both the significance of urmylation and Urm1 target identity are open issues in Saccharomyces cerevisiae. Nonetheless, based on the recent isolation of various human URM1 targets, assigning biological functions to protein urmylation seems feasible. Therefore, we propose two complementary strategies for studying Urm1 pathways in yeast. In the first, bioinformatics will identify selected yeast homologs of human targets for direct urmylation and cross-complementation assays. This has the potential to identify bona fide Urm1 targets and study conservation within Urm1 modification pathways among eukaryotes. The second uses a TAP-tagged bait of Urm1 for proteome-wide Urm1 target identification in yeast by mass spectrometry. To validate urmylation, candidates from each approach will be assessed by independent Urm1 conjugation assays and bona fide Urm1 interactions are correlated with phenotypic profiles obtained from strains mutated in the appropriate Urm1 target genes. The latter holds promise to ask how urmylation may affect target protein function and importantly, whether protein urmylation and tRNA thiolation represent related or independent branches of Urm1 modification pathways. In sum, by combining molecular, biochemical and bioinformatical methods, the proposal is likely to provide new insights into the biology and significance of the eukaryal Urm1 modification pathway.