Overlap of NatA and IAP substrates implicates N-terminal acetylation in protein stabilization

Müller F, Friese A, Pathe C, Da Silva RC, Rodriguez KB, Musacchio A, Bange T (2021) Science Advances 


SMAC/DIABLO and HTRA2 are mitochondrial proteins whose amino-terminal sequences, known as inhibitor of apoptosis binding motifs (IBMs), bind and activate ubiquitin ligases known as inhibitor of apoptosis proteins (IAPs), unleashing a cell’s apoptotic potential. IBMs comprise a four-residue, loose consensus sequence, and binding to IAPs requires an unmodified amino terminus. Closely related, IBM-like N termini are present in approximately 5% of human proteins. We show that suppression of the N-alpha-acetyltransferase NatA turns these cryptic IBMlike sequences into very efficient IAP binders in cell lysates and in vitro and ultimately triggers cellular apoptosis. 
Thus, amino-terminal acetylation of IBM-like motifs in NatA substrates shields them from IAPs. This previously unrecognized relationship suggests that amino-terminal acetylation is generally protective against protein degradation in human cells. It also identifies IAPs as agents of a general quality control mechanism targeting unacetylated rogues in metazoans.

Free Nt-α-amino forms of Nt-acetylated proteins bind to IAPs in cells. Left: Venn diagram summarizing numbers and percentages of all proteins with identified N termini from a large-scale MS experiment using HeLa cells. Total numbers and percentage of acetylated, dimethylated (reflecting unmodified, mono, and potentially also dimethylated N termini in cells), and proteins identified with both modifications are shown. Right: Scheme of the performed peptide pull-down experiments. Nt-Ac or free Nt-α-amino (Nt-free) biotinylated peptides were bound to streptavidin beads. Beads were then incubated with HeLa lysates, washed, and directly digested on beads for MS analysis. Quantification was performed by using label-free intensities (label-free quantitation) or stable isotope labeling by amino acids in cell culture (SILAC).
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