Fig. 3: Unacetylated hydrophobic NatC substrates are less stable.

a N-terminal variants of UBE2M-FLAG were transiently expressed in HAP1 NAA30-KO cells and protein levels were determined by immunoblotting (n = 3 independent experiments). The native N-terminus of UBE2M starts with MI. b HAP1 WT and NAA30-KO cells were transfected with the indicated UBE2M-V5-P2A-GST-GFP reporter construct, and protein levels were determined by immunoblot analysis. UBE2M-V5 levels were normalized to GST-GFP and expressed relative to WT sample. Data are shown as mean ± SD of four independent experiments. ***p = 0.0004; two-tailed unpaired t test. c NAA30-WT-V5 and NAA30-mut-V5 (E321A) was immunoprecipitated from HeLa cell extracts and used in Nt-acetylation assays with [¹⁴C]-acetyl-CoA and synthetic peptides representing the NatC substrates UBE2M (MIKL) and ARFRP1 (MYTL), and the NAA80/NatH substrate β-actin (DDDI). The experiment was performed three independent times with three technical replicates each. Data from one representative setup is shown as mean ± SD. DPM: disintegrations per minute. d NatC regulates the protein level of UBE2M, UBE2F, ARFRP1, and CAPNS1. Immunoblot analysis of HAP1 WT and NAA30-KO cells transfected with control V5 plasmid, NAA30-V5 or the catalytically dead mutant NAA30-mut-V5 (n = 3 biologically independent samples). e HAP1 WT and NAA30-KO cells were treated with proteasomal [MG132 and bortezomib (BMZ)] and lysosomal inhibitors [bafilomycin A (BafA), leupeptin (LP) or ammonium chloride (NH₄Cl)] for 6 h followed by immunoblot analysis using the indicated antibodies (n = 3 independent experiments). DMSO served as vehicle control. Source data are provided as a Source Data file.