The carnitine/acylcarnitine transporter (CACT; SLC25A20) mediates an antiport 
reaction allowing entry of acyl moieties in the form of acylcarnitines into the 
mitochondrial matrix and exit of free carnitine. The transport function of CACT 
is crucial for the β-oxidation pathway. In this work, it has been found that 
CACT is partially acetylated in rat liver mitochondria as demonstrated by 
anti-acetyl-lys antibody immunostaining. Acetylation was reversed by the 
deacetylase Sirtuin 3 in the presence of NAD+. After treatment of the 
mitochondrial extract with the deacetylase, the CACT activity, assayed in 
proteoliposomes, increased. The half-saturation constant of the CACT was not 
influenced, while the V max was increased by deacetylation. Sirtuin 3 was not 
able to deacetylate the CACT when incubation was performed in intact mitoplasts, 
indicating that the acetylation sites are located in the mitochondrial matrix. 
Prediction on the localization of acetylated residues by bioinformatics 
correlates well with the experimental data. Recombinant CACT treated with 
acetyl-CoA was partially acetylated by non-enzymatic mechanism with a 
corresponding decrease of transport activity. The experimental data indicate 
that acetylation of CACT inhibits its transport activity, and thus may 
contribute to the regulation of the mitochondrial β-oxidation pathway.