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

Abstract

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 N6-theronylcarbamoyl adenosine (t6A), 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. Mutations in KEOPS genes in humans underlie the severe genetic disease Galloway-Mowat syndrome, which results in childhood death. KEOPS activity depends on two major functions of Bud32. Firstly, Bud32 facilitates efficient tRNA substrate recruitment to KEOPS and helps in positioning the A37 site of the tRNA in the active site of Kae1, which carries out the t6A modification reaction. Secondly, the enzymatic activity of Bud32 is required for the ability of KEOPS to modify tRNA. Unlike conventional protein kinases, which employ their enzymatic activity for phosphorylation of protein substrates, Bud32 employs its enzymatic activity to function as an ATPase. Herein, we present a comprehensive suite of assays to monitor the activity of Bud32 in KEOPS in vitro and in vivo. We present protocols for the purification of the archaeal KEOPS proteins and of a tRNA substrate, as well as protocols for monitoring the ATPase activity of Bud32 and for analyzing its role in tRNA binding. We further present a complementary protocol for monitoring the role Bud32 has in cell growth in yeast.