Nt-acetylation-independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases

• Published in: Plant physiology (Bethesda) 2023-10, Vol.193 (3), p.2086-2104
• Main Authors: Etherington, Ross D, Bailey, Mark, Boyer, Jean-Baptiste, Armbruster, Laura, Cao, Xulyu, Coates, Juliet C, Meinnel, Thierry, Wirtz, Markus, Giglione, Carmela, Gibbs, Daniel J
• Format: Article
• Language: English
• Subjects: Biochemistry; Biochemistry, Molecular Biology; Botanics; Genomics; Life Sciences; Vegetal Biology
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1093/plphys/kiad406

Abstract The acetylation-dependent (Ac/)N-degron pathway degrades proteins through recognition of their acetylated N-termini (Nt) by E3 ligases called Ac/N-recognins. To date, specific Ac/N-recognins have not been defined in plants. Here we used molecular, genetic, and multiomics approaches to characterize potential roles for Arabidopsis (Arabidopsis thaliana) DEGRADATION OF ALPHA2 10 (DOA10)-like E3 ligases in the Nt-acetylation-(NTA)-dependent turnover of proteins at global- and protein-specific scales. Arabidopsis has two endoplasmic reticulum (ER)-localized DOA10-like proteins. AtDOA10A, but not the Brassicaceae-specific AtDOA10B, can compensate for loss of yeast (Saccharomyces cerevisiae) ScDOA10 function. Transcriptome and Nt-acetylome profiling of an Atdoa10a/b RNAi mutant revealed no obvious differences in the global NTA profile compared to wild type, 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 showed that turnover of ER-localized SQUALENE EPOXIDASE 1 (AtSQE1), a critical sterol biosynthesis enzyme, is mediated by AtDOA10s. Degradation of AtSQE1 in planta did not depend on NTA, but Nt-acetyltransferases indirectly impacted 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. Global- and protein-specific analyses reveal that Arabidopsis DOA10A-like E3 ligases regulate the turnover of SQUALENE EPOXIDASE 1 independently of the Ac/N-degron pathway.