Methods in Enzymology

Available online 9 July 2025
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Methods in Enzymology

Identification of the Ac/N-degron recognition domain in the MARCHF6 E3 ubiquitin ligase

https://doi.org/10.1016/bs.mie.2025.06.027Get rights and content

Abstract

Nα-terminal (Nt-) acetylation is a prevalent post-translational modification that regulates protein stability and turnover. The Ac/N-degron pathway, a branch of the N-degron pathways, recognizes Nt-acetyl groups as degradation signals (Ac/N-degrons), mediating proteolysis. MARCHF6, an endoplasmic reticulum (ER)-transmembrane E3 ubiquitin ligase, acts as a principal Ac/N-recognin, targeting Ac/N-degron-bearing substrates for polyubiquitylation and subsequent proteasomal degradation. However, the molecular mechanisms underlying Ac/N-degron recognition by MARCHF6 remain elusive. Here, we utilized a comprehensive alanine-stretch mutational screen combined with split-ubiquitin (Split-Ub) assays to define the Ac/N-degron recognition domain (Ac/N-domain) within MARCHF6. Sequence alignment with its yeast ortholog, Doa10, revealed conserved cytosolic residues essential for substrate recognition. Biochemical approaches, including chemical crosslinking and co-immunoprecipitation, identified key residues critical for Ac/N-degron recognition, while truncation and Split-Ub assays delineated the specific Ac/N-domain necessary for binding. These findings establish a mechanistic framework for Ac/N-degron recognition by MARCHF6, deepening our understanding of Nt-acetylation-mediated proteostasis and its therapeutical implications for diseases linked to dysregulation of Nt-acetylation or MARCHF6, including cancer, birth defects, and metabolic and neurological disorders.

Introduction

Nα-terminal (Nt-) acetylation is a ubiquitous and evolutionarily conserved post-translational modification in eukaryotic cells, occurring either co- or post-translationally in the majority of cellular proteins. This modification influences protein folding, stability, turnover, subcellular localization, and molecular interactions (Calis and Gevaert, 2025, McTiernan et al., 2025).
The N-degron pathways, formerly known as the N-end rule pathways, are conserved proteolytic systems that regulate protein degradation by recognizing specific Nt-residues as degradation signals (N-degrons) (Heo et al., 2023, Varshavsky, 2024). Among these, the Ac/N-degron pathway selectively targets Nt-acetylated proteins, using their acetylated N-termini as degradation signals (Ac/N-degrons) for polyubiquitylation-mediated proteasomal degradation (Hwang et al., 2010, Nguyen et al., 2018, Yang and Hwang, 2024).
MARCHF6, an endoplasmic reticulum (ER)-transmembrane E3 ubiquitin (Ub) ligase, serves as the principal Ac/N-recognin of the Ac/N-degron pathway (Varshavsky, 2024, Yang et al., 2023). This multi-spanning transmembrane enzyme consists of 14 transmembrane helices and eight cytosolic regions, including an N-terminal catalytic RING domain and a NADPH-sensing C-terminal regulatory MRR domain (Nguyen et al., 2022, Yang et al., 2023). MARCHF6 mediates the degradation of Ac/N-degron-bearing substrates such as the G-protein regulator RGS2 and the lipid droplet-associated protein PLIN2 (Nguyen et al., 2019, Park et al., 2015). Additionally, MARCHF6 targets, for degradation, non-Ac/N-degron-bearing proteins such as the tumor suppressor p53, the long-chain acyl-CoA synthase ACSL4, and cytosolically mislocalized proopiomelanocortin (POMC)—thereby modulating ferroptosis, an iron-dependent lipid peroxidation-driven mode of regulated cell death (Berndt et al., 2024, Mun and Hwang, 2024, Mun et al., 2023, Nguyen et al., 2022, Yang et al., 2024). MARCHF6 also regulates cholesterol homeostasis by degrading squalene monooxygenase, independent of Nt-acetylation (Scott et al., 2021, Yang et al., 2024).
Despite significant progress in defining the molecular architecture and cellular functions of MARCHF6 and its orthologs, yeast Doa10 and plant Doa10-like proteins (Botsch et al., 2024, Etherington et al., 2023, Mehrtash and Hochstrasser, 2022, Wu et al., 2024), the precise molecular determinants governing Ac/N-degron recognition remain unclear. This knowledge gap stems from several challenges: (1) the inherent biochemical complexity of ER-resident transmembrane proteins, (2) limited structural information on the cytosol-facing unstructured regions of MARCHF6, and (3) the intricate interplay between Ac/N-degrons and non-Ac/N-degrons, which may function either exclusively or cooperatively depending on the Nt-acetylation status of the target protein (Scott et al., 2021, Yang and Hwang, 2024, Yang et al., 2024). Resolving these questions is critical for advancing our understanding of the Ac/N-degron pathway and its broader implications in proteostasis regulation.
Given that Nt-acetylation predominantly occurs in the cytosol (Calis and Gevaert, 2025, McTiernan et al., 2025), we hypothesized that MARCHF6 recognizes Ac/N-degron substrates through a cytosol-facing domain. To systematically identify this domain, we employed two complementary approaches: (1) alanine-scanning mutagenesis targeting evolutionarily conserved residues within the cytosolic domains of MARCHF6, followed by biochemical analyses using chemical crosslinking and co-immunoprecipitation (Co-IP); and (2) split-ubiquitin (Split-Ub) assays with truncated MARCHF6 fragments and isolated cytosolic domains to delineate the Ac/N-degron recognition region (Fig. 1) (Yang et al., 2024). Through these methodologies, we aimed to define the Ac/N-domain within MARCHF6, providing mechanistic insights into its role in regulated protein degradation and its rheostatic function in ferroptosis (Yang et al., 2024). The subsequent sections detail the methodologies employed in this study.

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Section snippets

Systematic alanine-stretch mutational screening to identify the Ac/N-degron recognition region in MARCHF6

To determine the cytosolic regions of MARCHF6 involved in Ac/N-degron recognition, we performed a systematic alanine-stretch mutational screening. Sequence alignment of MARCHF6 from humans and mice, along with its yeast ortholog Doa10, revealed clusters of evolutionarily conserved residues within its eight cytosol-facing regions. Based on this analysis, we generated 25 MARCHF6 variants by substituting consecutive stretches of three to five conserved residues with alanine. These variants were

Mapping Ac/N-degron binding sites in MARCHF6 using the Split-Ub technique

To further define the Ac/N-degron binding site within MARCHF6, we utilized the split-Ub system (Möckli et al., 2007). This technique detects protein-protein interactions by reconstituting ubiquitin upon proximity, leading to cleavage by deubiquitinating enzymes (DUBs) and activation of a reporter.
We applied two complementary strategies:
  • 1.
    C-terminally truncated MARCHF6 constructs to assess whether multiple regions contribute to Ac/N-degron recognition (Fig. 4A and B).
  • 2.
    Individually expressed

Summary and conclusion

Evolutionary conservation analysis, systematic alanine stretch-mutational screening, Split-Ub assays, and biochemical approaches, including chemical crosslinking and co-immunoprecipitation, identified a critical Ac/N-domain in the fifth cytosol-facing region of MARCHF6 essential for Ac/N-degron recognition. Cycloheximide chase assays confirmed its functional significance in selectively degrading Ac/N-degron substrates, while mutations in this domain impaired substrate recognition, increasing

Acknowledgments

Fig. 3, Fig. 4, Fig. 5 are reprinted with permission from Elsevier (Yang et al., 2024). This study was supported by the Korean government (MSIP) grant, NRF-2020R1A3B2078127 and Korea University grant, K2502851. We thank both current and former members of Hwang laboratory for their valuable advice and assistance.

Closure statement

During the preparation of this manuscript, the authors utilized ChatGPT-4.0 (OpenAI) for assistance with English language editing. Following this, the authors reviewed and edited the

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