Abstract
AbstractSIRT4 comprises together with SIRT3 and SIRT5 the mitochondrially localized subgroup of sirtuins. SIRT4 regulatesviaits NAD+-dependent enzymatic activities mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy). Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl2-induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, within the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4-eGFP decrease upon CoCl2treatment 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 CoCl2-induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl2treatment 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, (i)viamitochondrial autophagy/mitophagy, and (ii) as a separate processviaproteasomal degradation within the cytoplasm.
Publisher
Cold Spring Harbor Laboratory