Fig. 1.

NAA80 Nt-acetylates β- and γ-actin. (A) NAA80-V5 was immunoprecipitated from HeLa cells and used in Nt-acetylation assays with [¹⁴C]-Ac-CoA and peptides representing a variety of N termini. (B) NAA80-eGFP expressed in HeLa and HAP1 cells shows a cytosolic distribution. (C) Phosphorescence imaging shows a distinct 43-kDa band in NAA80 KO1 cell lysates incubated with [¹⁴C]-Ac-CoA and purified recombinant MBP-NAA80, suggesting the incorporation of [¹⁴C]-Ac at the N terminus of actin. (D) The isoform and Nt-acetylation specificity of Ac-β-actin and Ac-γ-actin antibodies is confirmed in dot blot assays with peptides representing the unacetylated and acetylated N termini of β- and γ-actin. (E) NAA80 expression is required for actin Nt-acetylation. Immunoblot analysis of HAP1 control and NAA80 KO1 cells transfected with empty V5 plasmid, wild-type NAA80-V5, or the catalytically inactive mutant NAA80mut-V5. The samples were probed with Ac-β-actin, Ac-γ-actin, pan-actin, V5, and GAPDH antibodies. (F) Actin acetylation is eliminated in NAA80-KO cells and restored upon reexpression of active NAA80. HAP1 control cells were transfected with an empty eGFP vector, and NAA80 KO1 cells were additionally transfected with NAA80-eGFP or NAA80mut-eGFP and stained with Ac-β-actin or Ac-γ-actin antibodies and analyzed by IF. The general presence of F-actin in the analyzed cells was visualized by Rhodamine phalloidin staining (Bottom Row). Of note, the cells shown here are from separate samples not subjected to antibody labeling. (Scale bars, 10 µm.)