Sua5 catalyzing universal t 6 A tRNA modification is responsible for multifaceted functions of the KEOPS complex in Cryptococcus neoformans

The N 6 -threonylcarbamoyl adenosine (t 6 A) tRNA modification is critical for ensuring translation fidelity across three domains of life. Our prior work highlighted the KEOPS complex, organized in a Pcc1-Kae1-Bud32-Cgi121 linear arrangement, not only serves an evolutionarily conserved role in t 6 A tRNA modification but also exerts diverse functional impacts on pathobiological characteristics in Cryptococcus neoformans , a leading cause of fungal meningitis worldwide. However, the extent to which the pleiotropic functions of the KEOPS complex are specifically tied to tRNA modification remains uncertain. To address this, we undertook a functional characterization of Sua5, responsible for generating the precursor threonylcarbamoyl-adenylate (TC-AMP) for t 6 A tRNA modification, using a reverse genetics approach. Comparative phenotypic analyses with KEOPS mutants revealed that Sua5 plays a vital role in multiple cellular processes, such as t 6 A tRNA modification, growth, sexual development, stress response, and virulence factor production, thus reflecting the multifaceted functions of the KEOPS complex. In support of this, sua5 Δ bud32 Δ double mutants showed phenotypes comparable to those of the corresponding single mutants. Intriguingly, a SUA5 allele lacking a mitochondria targeting sequence ( SUA5 MTS Δ ) was sufficient to restore the wild-type phenotypes in the sua5 Δ mutant, suggesting that Sua5’s primary functional locus may be cytosolic, akin to the KEOPS complex. Further supporting this, the deletion of Qri7, a mitochondrial paralog of Kae1, had no discernible phenotypic impact on C. neoformans . We concluded that cytosolic t 6 A tRNA modifications, orchestrated by Sua5 and the KEOPS complex, are central to the regulation of diverse pathobiological functions in C. neoformans . IMPORTANCE Understanding cellular functions at the molecular level is crucial for advancing disease treatments. Our research reveals a critical connection between the KEOPS complex and Sua5 in Cryptococcus neoformans , a ...