The KEOPS complex is an evolutionarily conserved protein complex in all three domains of life (Bacteria, Archaea, and Eukarya). In budding yeast Saccharomyces cerevisiae, the KEOPS complex (ScKEOPS) consists of five subunits, which are Kae1, Bud32, Cgi121, Pcc1, and Gon7. The KEOPS complex is an ATPase and is required for tRNA N6-threonylcarbamoyladenosine modification, telomere length maintenance, and efficient DNA repair. Here, recombinant ScKEOPS full complex and Kae1–Pcc1–Gon7 and Bud32–Cgi121 subcomplexes were purified and their biochemical activities were examined. KEOPS was observed to have ATPase and GTPase activities, which are predominantly attributed to the Bud32 subunit, as catalytically dead Bud32, but not catalytically dead Kae1, largely eliminated the ATPase/GTPase activity of KEOPS. In addition, KEOPS could hydrolyze ADP to adenosine or GDP to guanosine, and produce PPi, indicating that KEOPS is an ADP/GDP nucleotidase. Further mutagenesis characterization of Bud32 and Kae1 subunits revealed that Kae1, but not Bud32, is responsible for the ADP/GDP nucleotidase activity. In addition, the Kae1V309D mutant exhibited decreased ADP/GDP nucleotidase activity in vitro and shortened telomeres in vivo, but showed only a limited defect in t6A modification, suggesting that the ADP/GDP nucleotidase activity of KEOPS contributes to telomere length regulation.

中文翻译：

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酿酒酵母 KEOPS 复合体的 Kae1 具有 ADP/GDP 核苷酸酶活性

KEOPS 复合体是一种在所有三个生命领域（细菌、古细菌和真核生物）中进化保守的蛋白质复合体。在出芽酵母酿酒酵母中，KEOPS 复合体 (ScKEOPS) 由五个亚基组成，它们是 Kae1、Bud32、Cgi121、Pcc1 和 Gon7。KEOPS 复合物是一种 ATP 酶，是 tRNA N6-苏氨酰氨基甲酰腺苷修饰、端粒长度维持和有效 DNA 修复所必需的。在这里，纯化了重组 ScKEOPS 全复合物和 Kae1–Pcc1–Gon7 和 Bud32–Cgi121 亚复合物，并检测了它们的生化活性。观察到 KEOPS 具有 ATPase 和 GTPase 活性，这主要归因于 Bud32 亚基，因为催化死亡的 Bud32，但不是催化死亡的 Kae1，在很大程度上消除了 KEOPS 的 ATPase/GTPase 活性。此外，KEOPS 可以将 ADP 水解为腺苷或将 GDP 水解为鸟苷，并产生 PPi，表明 KEOPS 是一种 ADP/GDP 核苷酸酶。Bud32 和 Kae1 亚基的进一步诱变表征表明，Kae1 而不是 Bud32 负责 ADP/GDP 核苷酸酶活性。此外，Kae1V309D 突变体表现出体外 ADP/GDP 核苷酸酶活性降低和体内端粒缩短，但在 t6A 修饰中仅表现出有限的缺陷，表明 KEOPS 的 ADP/GDP 核苷酸酶活性有助于端粒长度调节。