Wu Pengju, Gan Qi, Zhang Xuemei, Yang Yunfeng, Xiao Yuanxi, She Qunxin, Ni Jinfeng, Huang Qihong, Shen Yulong
State Key Laboratory of Microbial Technology, CRISPR and Archaea Biology Research Center, Microbial Technology Institute Shandong University Qingdao China.
mLife. 2023 Jan 8;2(1):11-27. doi: 10.1002/mlf2.12051. eCollection 2023 Mar.
Kinase, putative Endopeptidase, and Other Proteins of Small size (KEOPS) is a multisubunit protein complex conserved in eukaryotes and archaea. It is composed of Pcc1, Kae1, Bud32, Cgi121, and Gon7 in eukaryotes and is primarily involved in N-threonylcarbamoyl adenosine (tA) modification of transfer RNAs (tRNAs). Recently, it was reported that KEOPS participates in homologous recombination (HR) repair in yeast. To characterize the KEOPS in archaea (aKEOPS), we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon . We show that aKEOPS also possesses five subunits, Pcc1, Kae1, Bud32, Cgi121, and Pcc1-like (or Gon7-like), just like eukaryotic KEOPS. Pcc1-like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex (Kae1-Pcc1-Pcc1-Kae1), suggesting that Pcc1-like is a functional homolog of the eukaryotic Gon7 subunit. Strikingly, none of the genes encoding aKEOPS subunits, including Pcc1 and Pcc1-like, can be deleted in the wild type and in a tA modification complementary strain named TsaKI, implying that the aKEOPS complex is essential for an additional cellular process in this archaeon. Knock-down of the Cgi121 subunit leads to severe growth retardance in the wild type that is partially rescued in TsaKI. These results suggest that aKEOPS plays an essential role independent of the cellular tA modification level. In addition, archaeal Cgi121 possesses dsDNA-binding activity that relies on its tRNA 3' CCA tail binding module. Our study clarifies the subunit organization of archaeal KEOPS and suggests an origin of eukaryotic Gon7. The study also reveals a possible link between the function in tA modification and the additional function, presumably HR.
激酶、假定的内肽酶及其他小尺寸蛋白（KEOPS）是一种在真核生物和古细菌中保守的多亚基蛋白复合物。在真核生物中，它由Pcc1、Kae1、Bud32、Cgi121和Gon7组成，主要参与转运RNA（tRNA）的N-苏氨甲酰腺苷（tA）修饰。最近，有报道称KEOPS参与酵母中的同源重组（HR）修复。为了表征古细菌中的KEOPS（aKEOPS），我们对嗜热古菌中其编码基因进行了遗传和生化分析。我们发现，aKEOPS同样拥有五个亚基，即Pcc1、Kae1、Bud32、Cgi121和类Pcc1（或类Gon7），这与真核生物的KEOPS类似。类Pcc1与Kae1和Pcc1存在物理相互作用，并能介导二聚体亚复合物（Kae1-Pcc1-Pcc1-Kae1）的单体化，这表明类Pcc1是真核生物Gon7亚基的功能同源物。引人注目的是，包括Pcc1和类Pcc1在内的编码aKEOPS亚基的基因，在野生型和一个名为TsaKI的tA修饰互补菌株中均无法被敲除，这意味着aKEOPS复合物对于该古细菌中的另一个细胞过程至关重要。敲低Cgi121亚基会导致野生型中严重的生长迟缓，而在TsaKI中这种情况会得到部分缓解。这些结果表明，aKEOPS发挥着独立于细胞tA修饰水平的重要作用。此外，古细菌的Cgi121具有依赖于其tRNA 3' CCA尾结合模块的双链DNA结合活性。我们的研究阐明了古细菌KEOPS的亚基组成，并提示了真核生物Gon7的起源。该研究还揭示了tA修饰功能与另一个可能是HR的功能之间的潜在联系。
