Global and protein-specific analysis reveals Nt-acetylation-independent turnover of SQE1 by Arabidopsis DOA10-like E3 ligases

The Acetylation-dependent (Ac/) N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by specific E3-ligases (Ac/N-recognins). To date, no Ac/N-recognins have been defined in plants. Here we use molecular, genetic, and multi-omics approaches to characterise potential roles for Arabidopsis DOA10-like E3-ligases in the Nt-acetylation-(NTA-) dependent turnover of proteins at global and protein-specific scales. Arabidopsis has two ER-localised DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no significant differences in the global NTA profile compared to wildtype, suggesting that AtDOA10s do not regulate the bulk turnover of NTA substrates. Using protein steady-state and cycloheximide-chase degradation assays in yeast and Arabidopsis, we show that turnover of ER-localised squalene epoxidase 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta does not depend on NTA, but Nt-acetyltransferases indirectly impact its turnover in yeast, indicating kingdom-specific differences in NTA and cellular proteostasis. Our work suggests that, in contrast to yeast and mammals, targeting of Nt-acetylated proteins is not a major function of DOA10-like E3 ligases in Arabidopsis, and provides further insight into plant ERAD and the conservation of regulatory mechanisms controlling sterol biosynthesis in eukaryotes.