CoCl -triggered pseudohypoxic stress induces proteasomal degradation of SIRT4 via polyubiquitination of lysines K78 and K299.

SIRT4, together with SIRT3 and SIRT5, comprises the mitochondrially localized subgroup of sirtuins. SIRT4 regulates mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy) via its NAD -dependent enzymatic activities. Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl -induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, we observed that of the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4-eGFP decrease upon CoCl treatment of HEK293 cells. Co-treatment with BafA1, an inhibitor of autophagosome-lysosome fusion required for autophagy/mitophagy, or the use of the proteasome inhibitor MG132, prevented CoCl -induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl treatment and were more resistant to pseudohypoxia-induced degradation as compared to SIRT4. Moreover, SIRT4(K78R) and SIRT4(K299R) displayed increased basal protein stability as compared to wild-type SIRT4 when subjected to MG132 treatment or cycloheximide (CHX) chase assays. Thus, our data indicate that stress-induced protein degradation of SIRT4 occurs through two mechanisms: (a) via mitochondrial autophagy/mitophagy, and (b) as a separate process via proteasomal degradation within the cytoplasm.