激酶,推定的内肽酶,和其他小尺寸蛋白质(KEOPS)是真核生物和古细菌中保守的多亚基蛋白质复合物。它由真核生物中的Pcc1,Kae1,Bud32,Cgi121和Gon7组成,主要参与转移RNA(tRNA)的N6-苏酰基氨基甲酰基腺苷(t6A)修饰。最近,据报道,KEOPS参与酵母的同源重组(HR)修复。为了表征古细菌(AKEOPS)中的KEOPS,我们在超嗜热古细菌中对其编码基因进行了遗传和生化分析。我们证明AKEOPS还拥有五个亚基,Pcc1、Kae1、Bud32、Cgi121和Pcc1样(或Gon7样),就像真核细胞一样。Pcc1样与Kae1和Pcc1具有物理相互作用,并且可以介导二聚体亚复合物(Kae1-Pcc1-Pcc1-Kae1)的单体化,表明Pcc1样是真核生物Gon7亚基的功能同源物。引人注目的是,没有编码AKEOPS亚基的基因,包括Pcc1和Pcc1样,可以在野生型和名为TsaKI的T6A修饰互补菌株中删除,暗示aKEOPS复合体对于该古细菌中的额外细胞过程至关重要。Cgi121亚基的敲除在野生型中导致严重的生长迟缓,在TsaKI中部分被挽救。这些结果表明aKEOPS发挥重要作用,而与细胞t6A修饰水平无关。此外,古细菌Cgi121具有依赖于其tRNA3'CCA尾结合模块的dsDNA结合活性。我们的研究阐明了古细菌KEOPS的亚基组织,并暗示了真核生物Gon7的起源。该研究还揭示了t6A修改中的功能与附加功能之间的可能联系,大概HR。
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 N6-threonylcarbamoyl adenosine (t6A) 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 Saccharolobus islandicus. 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 t6A 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 t6A 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 t6A modification and the additional function, presumably HR.