A suite of in vitro and in vivo assays for monitoring the activity of the pseudokinase Bud32.

Bud32 is a member of the protein kinase superfamily that is invariably conserved in all eukaryotic and archaeal organisms. In both of these kingdoms, Bud32 forms part of the KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) complex together with the three other core subunits Kae1, Cgi121 and Pcc1. KEOPS functions to generate the universal and essential tRNA post-transcriptional modification N-theronylcarbamoyl adenosine (tA), which is present at position A37 in all tRNAs that bind to codons with an A in the first position (ANN decoding tRNAs) and is essential for the fidelity of translation.

A substrate binding model for the KEOPS tRNA modifying complex.

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality.