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. 2023 Oct 27;14:6774. doi: 10.1038/s41467-023-42342-y

Fig. 4. UBR4-KCMF1 targets unacetylated N-terminal methionine followed by a hydrophobic residue.

Fig. 4

a, b The protein level of endogenous UBE2M in HAP1 WT and NAA30-KO cells transfected with the indicated siRNAs for 72 h was assessed by immunoblotting (n = 4 biologically independent samples). c Schematic representation of protein capture by peptide pulldown. A set of 11-mer peptides derived from the N-terminal sequence of UBE2M were C-terminally labeled with K-biotin (MIKLFSLKQQK(K-biotin)) and conjugated to streptavidin magnetic beads. The first two residues were replaced to represent different N-termini (XY-UBE2M). Biotinylated UBE2M peptides were incubated with cell extracts and the pulled-down proteins were identified by immunoblot analysis. d In vitro peptide pulldown assay of UBR4-V5 transiently expressed in HeLa cells using acetylated and non-Nt-acetylated UBE2M peptide. e XY-UBE2M peptide pulldown assay with UBE2M peptides bearing different N-terminal amino acids and UBR4-V5 expressed in HeLa cells. f In vitro peptide pulldown assay of UBR4-N-FLAG expressed in HAP1 WT cells using acetylated and non-Nt-acetylated UBE2M peptide and X-nsP4 controls peptides. The UBR4-N construct contains the UBR-box (yellow), which is the substrate recognition domain of the UBR proteins. *indicates saturated UBE2A band. g HAP1 WT and NAA30-KO cells were transfected with siCtrl or siUBR4 for 72 h and protein abundance was determined by tandem mass tag (TMT)-based quantitative proteomics (see Supplementary Data 5; n = 4 biologically independent samples). Intensity profile plot showing protein levels of the top 100 proteins with abundance profiles most similar to UBE2M (blue trace) (one-way ANOVA, permutation-based FDR = 0.01, S0 = 0). The intensity profiles of RGS10, ARLB, CAPSN1, DIS3, and HK2 are highlighted in orange. h UBR4 knockdown stabilizes the protein levels of non-Nt-acetylated RGS10, HK1, DIS3, UBE2F, ARFRP1 and CAPNS1 in NAA30-KO cells. HAP1 WT and NAA30-KO cells were transfected with siCtrl or siUBR4 for 72 h followed by immunoblotting using the indicated antibodies (d–f and h; n = 3 independent experiments). Source data are provided as a Source Data file.