Phosphorylation of the α subunit inhibits proteasome assembly and regulates cell cycle in an archaeon

Archaea of the order Sulfolobales possess a eukaryote-like cell division machinery and display a eukaryote-like cell cycle; however, the cell division and cell-cycle control mechanisms remain enigmatic. Here, we demonstrate that phosphorylation of the α subunit by a eukaryote-like protein kinase, ePK2, affects 20S proteasome assembly and controls cell division in Saccharolobus islandicus. ePK2 exhibits cell-cycle-dependent expression at both transcriptional and translational levels. Deletion or overexpression of epk2 results in impaired cytokinesis, with the deletion cells being unable to generate single chromosome cells after synchronization and the overexpression cells exhibiting growth retardation and cell enlargement. Interestingly, overexpression of ePK2 leads to a coherent reduction in cellular proteasome activity and degradation of cell division proteins. We identify S200 and T213 of the proteasome α subunit as specific target sites for ePK2 phosphorylation. Functional analyses of site-directed mutants at S200 and T213 suggest that phosphorylation at these two residues disrupts the assembly of de novo 20S proteasome. Collectively, our study uncovers an ingenious and efficient mechanism of proteasome phosphorylation-mediated cell division regulation, a prototype of the eukaryotic cell-cycle regulation system, in Sulfolobales archaea.