Acetylation of GOT2 at K159, K185, and K404 enhances its protein interaction with MDH2 A Cartoon representation of the malate–aspartate shuttle.Figure 1A Cartoon representation of the malate-aspartate shuttle. As shown, this shuttle is operated by two pairs of enzymes, cytosolic GOT1 and MDH1 as well as mitochondrial GOT2 and MDH2, which act in concert to transfer reducing equivalents across the mitochondrial membrane. B, C GOT2 can be acetylated. Flag‐tagged GOT2 was ectopically expressed in HEK293T cells treated with NAM (5 mM) and/or TSA (0.5 mM) for the indicated time period. Acetylation levels of Flag‐bead‐purified GOT2 were determined by Western blot analysis using a pan‐anti‐acetyllysine antibody (α‐Ac). IB and IP denote immunoblotting and immunoprecipitation, respectively. Relative GOT2 acetylation ratios were calculated after normalizing against Flag. D GOT2 acetylation enhances the interaction between ectopically expressed GOT2 and MDH2. Flag‐GOT2 and Myc‐MDH2 were co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐bead‐purified GOT2 and the protein association between ectopic proteins of GOT2 and MDH2 were determined by Western blot analysis. E Acetylation enhances the interaction between ectopically expressed GOT2 and endogenous MDH2. Flag‐GOT2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The acetylation level of Flag‐GOT2 and its association with endogenous MDH2 were determined by Western blot analysis. F Acetylation enhances the interaction between ectopically expressed MDH2 and endogenous GOT2. Flag‐MDH2 was co‐overexpressed in HEK293T cells treated without or with NAM (5 mM) for the indicated time period. The protein association between Flag‐MDH2 and endogenous GOT2 was determined by Western blot analysis. G Cartoon representation of GOT2 structure (PDB ID: 3PDB) (Han et al) made by Pymol (www.pymol.org). Three putative lysine residues (i.e., K159, K185, and K404) were labeled in red. See also Supplementary Fig S3. H Mapping the major lysine residue(s) of acetylation in GOT2 whose acetylation can affect protein interaction between GOT2 and MDH2. Putative acetylated residues were divided into six groups according to their position in the structure of GOT2. Each group of putative acetylated lysine (K) sites was mutated to arginine (R), and the deacetylated mimic K‐to‐R mutants were examined for their protein association with ectopically expressed Myc‐MDH2 by Western blot analysis. I GOT2 coupled with MDH2 shows a high level of K159 acetylation. Flag‐GOT2 was overexpressed in HEK293T cells without or with co‐overexpression of Myc‐MDH2. These transfected cells were treated with NAM (5 mM) for 5 h. Double immunoprecipitation was performed to obtain the Flag‐GOT2 from the Myc‐MDH2 immunoprecipitates (Ppt, supposed to be GOT2 bound with MDH2). The remaining supernatants (Sup, supposed to be GOT2 not bound with MDH2) were also harvested and precipitated by Flag antibody to test the K159 acetylation level of Flag‐GOT2 by Western blot analysis. J The 3KQ/R mutations do not change GOT2 enzyme activity. Wild‐type and 3K mutant GOT2 proteins were overexpressed and purified from E. coli, and the enzyme activity of GOT2 was determined as described in "". Shown are average values with standard deviation (SD) of triplicated experiments. n.s. = not significant for the indicated comparison.