Schwer and E
Schwer and E. HDM201 mitochondrial protein acetylation is markedly elevated in Sirt3?/? tissues. In addition, in the absence of Sirt3, multiple components of Complex I of the electron transport chain demonstrate increased acetylation. Sirt3 can also physically interact with at HDM201 least one of the known subunits of Complex I, the 39-kDa protein NDUFA9. Functional studies demonstrate that mitochondria from Sirt3?/? animals display a selective inhibition of Complex I activity. Furthermore, incubation of exogenous Sirt3 with mitochondria can augment Complex I activity. These results implicate protein acetylation as an important regulator of Complex I activity and demonstrate that Sirt3 functions to regulate and maintain basal ATP levels. ATP levels. Furthermore, we demonstrate that Sirt3 can reversibly bind to and regulate the acetylation and activity of Complex I of the electron-transport chain. Results The Sirt3 locus was inactivated by homologous recombination in ES cells leading to the deletion of exons 2C4 [supporting information (SI) Fig. S1]. Analysis of the tissues of homozygously deleted mice revealed undetectable levels of Sirt3 protein expression (Fig. S1). Consistent with a recent report (11), our own extensive survey of multiple organs by both gross and microscopic pathology revealed that Sirt3?/? mice appeared indistinguishable from wild-type littermates. In addition, we observed no increased or decreased mortality of these mice during the first year of life (H.-S.K. and C.-X.D., unpublished observations). In an effort to more fully understand the role of Sirt3 in mitochondrial biology, Sirt3+/? mice were bred to generate multiple independent cultures of wild-type or Sirt3?/? mouse embryonic fibroblasts (MEFs). Given the central role of mitochondria in generating ATP via the electron-transport chain (ETC), we first asked whether deletion of Sirt3 altered basal ATP levels. We measured ATP levels in five independent lines of wild-type MEFs and a similar number of independent Sirt3?/? MEFs (Fig. 1 0.01). We next asked whether transient reconstitution of Sirt3 could restore this ATP deficit. Sirt3?/? MEFs were transiently transfected with an empty vector, wild-type Sirt3, or a deacetylase-inactive form of Sirt3, Sirt3(HY). All transfections were performed with a cotransfected GFP expression vector to allow for subsequent purification of transfected cells using FACS sorting. As noted in Fig. 1= 3; mean SD). Shown is the corresponding level of expressed Sirt3 within each tissue as CACNB4 well as the 70-kDa complex II-associated protein Fp (CII-Fp) as a general measure of mitochondrial number and GAPDH for protein loading. (= 4 animals per group). *, 0.01. Based on these observations, we asked whether Sirt3 might also regulate ATP with deacteylase reaction HDM201 buffer only (? ) or with reaction buffer containing purified Sirt3 or Sirt4. The level of the Complex I protein NDUFA9 is shown as a loading control as is the level of exogenously added Flag-tagged Sirt3 HDM201 and Sirt4. (Complex I acetylation in HeLa cells transfected with empty vector (V), wild-type Sirt3 (WT), or a deacetylase inactive form of Sirt3 (HY). IP, immunoprecipitation; MW, molecular mass. Consistent with a role for mitochondrial sirtuins in regulating the ETC, treatment of HeLa cells with the broad-spectrum sirtuin inhibitor, nicotinamide, led to markedly enhanced Complex I acetylation (Fig. S3). In general, under basal conditions, the level of Complex I acetylation was higher in cultured human HeLa cells compared with isolated mouse liver mitochondria. We next asked whether Sirt3 could directly deacetylate Complex I. Nicotinamide-treated HeLa cells were used as a source to immunocapture Complex I that was then exposed for 2 h to either recombinant Sirt3 or Sirt4. Compared with samples exposed to Sirt4 or maintained in deacetylase buffer alone, Complex I incubated in the presence of exogenous Sirt3 protein demonstrated marked deacetylation of numerous proteins (Fig. 2oxidase (Complex IV) inhibitor, produced a similar magnitude reduction in ATP levels in wild-type and Sirt3?/? MEFs. Open in a separate window Fig. 3. Sirt3 associates with Complex I of the ETC. (and and and and.