Sua5 catalyzing universal t⁶A tRNA modification is responsible for multifaceted functions of the KEOPS complex in...

The N⁶-threonylcarbamoyl adenosine (t⁶A) 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 t⁶A 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 t⁶A 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 t⁶A tRNA modification, growth, sexual development, stress response, and virulence factor production, thus reflecting the multifaceted functions of the KEOPS complex. In support of this, sua5Δ bud32Δ double mutants showed phenotypes comparable to those of the corresponding single mutants. Intriguingly, a SUA5 allele lacking a mitochondria targeting sequence (SUA5^MTSΔ) was sufficient to restore the wild-type phenotypes in the sua5Δ 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 t⁶A tRNA modifications, orchestrated by Sua5 and the KEOPS complex, are central to the regulation of diverse pathobiological functions in C. neoformans.IMPORTANCEUnderstanding 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 significant cause of fungal meningitis. While the KEOPS complex is known for its versatile roles in cellular processes, Sua5 is specialized in t⁶A tRNA modification. Our key finding is that the diverse roles of the KEOPS complex, ranging from cell growth and stress response to virulence, are fundamentally linked to its function in t⁶A tRNA modification. This conclusion is supported by the remarkable similarities between the impacts of Sua5 and KEOPS on these processes, despite their roles in different steps of the t⁶A modification pathway. This newfound understanding deepens our insight into fungal biology and opens new avenues for developing potential therapies against dangerous fungal diseases.