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 shielding of the N-terminus stabilizes diverse cytosolic plant proteins. The Huntingtin yeast partner K (HYPK) facilitates NatA activity in planta, but in vitro, the N-terminal a-helix1 of HYPK inhibits NatA in the ternary NatA/HYPK complex. Consequently, the mechanism of HYPK function in plants was unknown. To understand the regulatory function of HYPK at the molecular level, we genetically engineered CRISPR/Cas9 mutants expressing either a non-functional HYPK (hypk-cr1) or an N-terminally deleted HYPK version (hypk-cr2). Like NatA-activity-depleted mutants, the hypk-cr1 and –cr2 mutants were more tolerant to drought stress and suffered from global proteome destabilization. Surprisingly, we found that absence of HYPK led to destabilization of the NatA subunits in leaves but not in roots of hypk-cr1. Further genetic and molecular evidence demonstrated that the stability of NatA subunits in leaves is mainly controlled by the C-terminal UBA domain of HYPK. While retaining the UBA domain, NatA activity was still impaired in hypk-cr2 leaves, implying that the N terminus of HYPK also contributes to the regulation of NatA activity. Furthermore, crossing of hypk-cr1 with a NatA-depleted mutant uncovered that HYPK promotes NatA activity not only by stabilizing NatA. Taken together, the activity-promoting function of the HYPK N-terminus depends on the binding of HYPK to NatA via the UBA domain, and is critical for NatA substrates starting with various amino acids. Our findings improve our understanding of the NatA regulatory subunit HYPK.