Molecular Mechanism and Physiological Functions of MARCH6 E3 Ubiquitin Ligase, a Key Recognition Component of the Ac/N-Degron Pathway

Abstract

Nα-terminal acetylation (Nt-acetylation) is one of the most prevalent protein modifications, which affects 50-90% of eukaryotic proteins. Although Nt-acetylation occurs ubiquitously, its general functions remain elusive. The discovery of the Ac/N-degron pathway that targets the Nt-acetyl group of proteins for degradation has shed a light on the unprecedented function of Nt-acetylation. In mammals, an endoplasmic reticulum (ER) transmembrane MARCH6 E3 ubiquitin (Ub) ligase, a homolog of yeast Doa10, is defined as an Nt-acetylation-recognition component (an Ac/N-recognin). Despite the prevalence of Nt-acetylation, a limited number of substrates of the Ac/N-degron pathway has been characterized thus far. Here, I identify perlipin2 (PLIN2), a major lipid droplet (LD)-associated protein, as a previously unknown substrate of the Ac/N-degron pathway. I find that MARCH6 specifically recognizes Nt-acetyl moiety of PLIN2 for its polyubiquitin-mediated and subsequent proteasome-dependent degradation. Using the modified split-Ub assay, I also show that the interaction strength of MARCH6 and PLIN2 depends on the tendency of Nt-acetylation of PLIN2. Additionally, I find that MARCH6 controls the amounts of intracellular LDs by degrading PLIN2, thereby revealing a previously unappreciated role of the Ac/N-degron pathway in lipid metabolism. On the other hand, my current works unveil the molecular mechanism of MARCH6 via its cytosolic C-terminal domain (CTD). Through split-Ub and GST pulldown assays, I demonstrate that CTD interacts specifically with the RING domain, a catalytic region of MARCH6. Specially, CTD drastically increases the catalytic activity of the RING by accelerating the transfer of Ub from an E2 (an Ub-conjugating enzyme thioesterified with Ub (E2∼Ub) to free Ub or poly-Ub chains. Interestingly, UBE2J2 and UBE2G2 E2s act together for the MARCH6-mediated poly-Ub chain formation: UBE2J2 preferentially contributes to short K48-linked Ub chains and UBE2G2 extends the poly-Ub chains from UBE2J2-established Ub chains. Furthermore, I find that MARCH6 senses cellular NADPH concentration for its catalytic activity.