Abstract
Squalene monooxygenase (SM) is a rate-limiting enzyme in cholesterol synthesis. The region comprising the first 100 amino acids, termed SM N100, represents the shortest cholesterol-responsive degron and enables SM to sense excess cholesterol in the endoplasmic reticulum (ER) membrane. Cholesterol accelerates the ubiquitination of SM by membrane-associated ring-CH type finger 6 (MARCH6), a key E3 ubiquitin ligase involved in ER-associated degradation. However, the ubiquitination site required for cholesterol regulation of SM N100 is unknown. Here, we used SM N100 fused to GFP as a model degron to recapitulate cholesterol-mediated SM degradation and show that neither SM lysine residues nor the N terminus impart instability. Instead, we discovered four serines (Ser-59, Ser-61, Ser-83, and Ser-87) that are critical for cholesterol-accelerated degradation, with MS analysis confirming Ser-83 as a ubiquitination site. Notably, these two clusters of closely spaced serine residues are located in disordered domains flanking a 12-amino acid-long amphipathic helix (residues Gln-62-Leu-73) that together confer cholesterol responsiveness. In summary, our findings reveal the degron architecture of SM N100, introducing the role of non-canonical ubiquitination sites and deepening our molecular understanding of how SM is degraded in response to cholesterol.
Highlights
Squalene monooxygenase (SM) is a rate-limiting enzyme in cholesterol synthesis
We have previously shown that SM N100 is degraded via the ubiquitin-proteasome system when excess cholesterol is present, even when all five lysine residues (Lys-15, Lys-16, Lys-82, Lys-90, and Lys-100) have been substituted with arginine [10]
We generated a construct where mCherry was introduced to sterically hinder the N terminus of SM N100 (Fig. 1A, right), impairing potential N-terminal ubiquitination. Introducing this bulky tag resulted in slight blunting (24%) of cholesterol regulation of SM N100 but no change in protein expression when comparing untreated conditions (Fig. 1, D and E)
Summary
Non-canonical ubiquitination of the cholesterol-regulated degron of squalene monooxygenase. We discovered four serines (Ser-59, Ser-61, Ser-83, and Ser-87) that are critical for cholesterol-accelerated degradation, with MS analysis confirming Ser-83 as a ubiquitination site. These two clusters of closely spaced serine residues are located in disordered domains flanking a 12-amino acid-long amphipathic helix (residues Gln-62–Leu-73) that together confer cholesterol responsiveness. We uncovered four key serine residues required for the cholesterol-accelerated degradation of SM N100 These serine sites are essential for degradation by membrane-associated ring-CH-type finger 6 (MARCH6), the E3 ubiquitin ligase for MARCH6, membrane-associated ring-CH-type finger 6; CD, methyl-cyclodextrin; DMEM, Dulbecco’s modified Eagle’s medium; CHO, Chinese hamster ovary; ESI, electrospray ionization
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