Characterization of the carboxy‐terminal domain of the <scp>GPN</scp> ‐loop <scp>GTPase</scp> Npa3 reveals an intrinsically disordered region and phosphorylation‐dependent regulation in the absence of <i>BUD27</i>

Yeast GPN-loop GTPase 1 (Npa3) is a member of the GPN-loop GTPase family involved in RNA polymerase II (RNAPII) assembly and nuclear targeting. Npa3 features a GTPase core and an enlarged carboxy-terminal domain (CTD) that is conserved in all eukaryotic orthologs but absent from the archaeal Gpn protein. We previously showed that Npa3 possesses additional cellular functions unrelated to RNAPII nuclear targeting. Although this enzyme was nuclear in cells expressing a truncated version of NPA3 lacking the last 106 residues (npa3∆C), these cells exhibited an increased sensitivity to the translation inhibitors hygromycin B and geneticin. Additionally, npa3∆C displayed a strong negative interaction with the bud site selection 27 deletion mutant (bud27∆). Despite the functional importance of the Npa3 CTD, little is known about its structure and regulation. Here, we show that the CTDs of Npa3 and its human ortholog GPN1 are predicted as intrinsically disordered regions (IDRs) containing three molecular recognition features (MoRFs). Both CTDs are disordered and acidic but differ widely in their primary sequences. Interestingly, Npa3 CTD function could be partially replaced by human GPN1 CTD. In proteomic studies, Npa3 was reported to be phosphorylated mainly at its CTD, but its physiological relevance remains unknown. In a bud27∆ background, strains expressing exclusively full-length, non-phosphorylatable Npa3, with eight CTD residues reported to be phosphorylated, mutated to alanine, displayed markedly increased sensitivity to hygromycin B and cycloheximide. The same phenotype was observed after simultaneously mutating only Ser304, Ser308, and Ser313 to alanine. We conclude that the Npa3 GTPase domain function is critically regulated by its disordered CTD through phosphorylation of the Ser304/Ser308/Ser313 cluster.