This study has established that murine SIRT3 is a soluble mitochondrial protein controlling global mitochondrial protein acetylation levels. SIRT3 represents the first factor known to affect protein acetylation in this compartment. In contrast, deletion of SIRT4 or SIRT5 is not associated with globally increased mitochondrial lysine acetylation, in agreement with previous reports showing that these factors possess little or no deacetylase activity (1
). We cannot exclude the possibility that SIRT4 and SIRT5 deacetylate specific mitochondrial factors not detected by our methodology. Surprisingly, despite accumulating hyperacetylated mitochondrial proteins, SIRT3-deficient mice are healthy under normal laboratory conditions and conditions of mild stress such as short-term food deprivation and show normal overall metabolism and cold resistance. We have focused on a metabolic characterization of SIRT3-deficient mice under basal conditions and conditions of mild nutritional stress, such as 24-h fasting; future studies are required to determine whether acute stressors such as oxidative insult, food deprivation, and conditions that require high levels of oxidative metabolism over a long period of time (e.g., high-fat diet) will uncover abnormalities in these mice. Along these lines, given that SIRT3 levels increase with CR in mice (33
), SIRT3 may regulate mitochondrial protein function in response to CR. Of interest, it has been reported that polymorphisms in the human SIRT3 gene are linked to longevity (5
). In this context, formal life span analysis of SIRT3-deficient mice under various dietary regimens will be needed to establish a firm role for this protein in regulating life span overall and the CR response in particular. Alternatively, in light of the central role of mitochondria in many cellular processes, SIRT3 might have functions aside from metabolism. For example, given the importance of mitochondria in the regulation of cell survival, a role for SIRT3 in apoptosis is conceivable.
We have identified GDH as one target of SIRT3. The functional significance of GDH acetylation is unclear; chemical acetylation of GDH has been shown to reduce its enzymatic activity in vitro (8
). Future studies will be required to further address the role of reversible lysine acetylation in GDH regulation. Notably, GDH is ADP-ribosylated by SIRT4, leading to decreased GDH activity and decreased insulin secretion in response to amino acids (19
). It will be of interest to determine whether SIRT3 and SIRT4 coregulate GDH and under which conditions this might occur. SIRT4 activity was proposed to decline with CR to permit higher levels of GDH activity (19
). CR is associated with elevated SIRT3 expression (33
) and potentially increased SIRT3 activity. Depending on the functional consequence of GDH acetylation, SIRT3 and SIRT4 may regulate GDH and potentially other proteins in a similar fashion during CR.
Given the pronounced hyperacetylation present in SIRT3-deficient mitochondria, the lack of an overt phenotype in these animals is puzzling. It is possible that, although mitochondrial protein hyperacetylation is dramatic in SIRT3 knockout mice at the level of immunoblot analysis, only a minor proportion of any particular factor is acetylated. Consequently, most proteins, e.g., metabolic enzymes such as GDH, would still be fully functional, explaining the absence of a strong metabolic phenotype in SIRT3-deficient mice. This possibility is supported by data showing that increased GDH acetylation in SIRT3-deficient mice does not result in grossly impaired GDH function, as indicated by normal tissue levels of the GDH substrate α-ketoglutarate (see Fig. S9 in the supplemental material). Presently, factors responsible for lysine acetylation of mitochondrial proteins are unknown. It might be necessary to overexpress the putative mitochondrial acetyltransferase(s) and/or delete putative redundant deacetylases in order to elicit a phenotype of SIRT3 deficiency. Indeed it is unclear whether lysine acetylation of mitochondrial proteins occurs in the mitochondrion itself or in the cytosol prior to mitochondrial import. Similarly, the biological function of mitochondrial protein acetylation is entirely unclear (21
). This modification could a priori regulate enzymatic activity, intramitochondrial localization, protein-protein interactions, protein stability, or some combination of these. Overall, SIRT3 represents the first factor described to affect global mitochondrial lysine acetylation, and SIRT3-deficient mice should serve as a valuable tool to study reversible lysine acetylation biology in mitochondria in vivo.