HYPK controls stability and catalytic activity of the N-terminal acetyltransferase A in Arabidopsis thaliana

The ribosome -tethered N -terminal acetyltransferase A (NatA) acetylates 52% of soluble proteins in Arabidopsis thaliana. This co -translational modification of the N terminus stabilizes diverse cytosolic plant proteins. The evolutionary conserved Huntingtin yeast partner K (HYPK) facilitates NatA activity in planta, but in vitro, its N -terminal helix o1 inhibits human NatA activity. To dissect the regulatory function of HYPK protein domains in vivo, we genetically engineer CRISPR-Cas9 mutants expressing a HYPK fragment lacking all functional domains (hypk-cr1) or an internally deleted HYPK variant truncating helix o1 but retaining the C -terminal ubiquitin-associated (UBA) domain (hypk-cr2). We find that the UBA domain of HYPK is vital for stabilizing the NatA complex in an organ -specific manner. The N terminus of HYPK, including helix o1, is critical for promoting NatA activity on substrates starting with various amino acids. Consequently, deleting only 42 amino acids inside the HYPK N terminus causes substantial destabilization of the plant proteome and higher tolerance toward drought stress.