Abstract
SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro, succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5-/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5-/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5-/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function.
Highlights
SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle
Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function
SIRT5 binds to cardiolipin on the inner mitochondrial membrane
Summary
Fractionated mitochondria from wild-type and SIRT5Ϫ/Ϫ mouse liver were subjected to Western blotting with anti-succinyllysine antibody Proteins in both the matrix and membrane fractions were observed to be hypersuccinylated in the absence of SIRT5 (Fig. 1A). Mass spectrometry identified 14 SIRT5 target sites on the SDHA subunit of Complex II and another eight on SDHB. Molecular modeling revealed that six of the eight SIRT5 target sites on SDHB orient toward the predicted membrane interface where SDHB interacts with SDHC/SDHD (Fig. 7, A and B) We speculate that these six positively charged lysines play a role in Complex II cardiolipin binding and proper conformation for electron transfer. This is unrelated to the known defect in mitochondrial FAO in SIRT5Ϫ/Ϫ mice because mice lacking the key FAO enzyme long-chain acyl-CoA dehydrogenase (LCAD) do not show the lactic acidosis phenotype
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