Mammalian acetate-dependent acetyl CoA synthetase 2 contains multiple protein destabilization and masking elements.

Besides contributing to anabolism, cellular metabolites serve as substrates or cofactors for enzymes and may also have signaling functions. Given these roles, multiple control mechanisms likely ensure fidelity of metabolite-generating enzymes. Acetate-dependent acetyl CoA synthetases (ACS) are de novo sources of acetyl CoA, a building block for fatty acids and a substrate for acetyltransferases. Eukaryotic acetate-dependent acetyl CoA synthetase 2 (Acss2) is predominantly cytosolic, but is also found in the nucleus following oxygen or glucose deprivation, or upon acetate exposure. Acss2-generated acetyl CoA is used in acetylation of Hypoxia-Inducible Factor 2 (HIF-2), a stress-responsive transcription factor. Mutation of a putative nuclear localization signal in endogenous Acss2 abrogates HIF-2 acetylation and signaling, but surprisingly also results in reduced Acss2 protein levels due to unmasking of two protein destabilization elements (PDE) in the Acss2 hinge region. In the current study, we identify up to four additional PDE in the Acss2 hinge region and determine that a previously identified PDE, the ABC domain, consists of two functional PDE. We show that the ABC domain and other PDE are likely masked by intramolecular interactions with other domains in the Acss2 hinge region. We also characterize mice with a prematurely truncated Acss2 that exposes a putative ABC domain PDE, which exhibits reduced Acss2 protein stability and impaired HIF-2 signaling. Finally, using primary mouse embryonic fibroblasts, we demonstrate that the reduced stability of select Acss2 mutant proteins is due to a shortened half-life, which is a result of enhanced degradation via a nonproteasome, nonautophagy pathway.