HYPK promotes the activity of the N α -acetyltransferase A complex to determine proteostasis of nonAc-X 2 /N-degron-containing proteins.

In humans, the Huntingtin yeast partner K (HYPK) binds to the ribosome-associated N α -acetyltransferase A (NatA) complex that acetylates ~40% of the proteome in humans and Arabidopsis thaliana . However, the relevance of Hs HYPK for determining the human N-acetylome is unclear. Here, we identify the At HYPK protein as the first in vivo regulator of NatA activity in plants . At HYPK physically interacts with the ribosome-anchoring subunit of NatA and promotes N α -terminal acetylation of diverse NatA substrates. Loss-of- At HYPK mutants are remarkably resistant to drought stress and strongly resemble the phenotype of NatA-depleted plants. The ectopic expression of Hs HYPK rescues this phenotype. Combined transcriptomics, proteomics, and N-terminomics unravel that HYPK impairs plant metabolism and development, predominantly by regulating NatA activity. We demonstrate that HYPK is a critical regulator of global proteostasis by facilitating masking of the recently identified nonAc-X 2 /N-degron. This N-degron targets many nonacetylated NatA substrates for degradation by the ubiquitin-proteasome system.