The E3 Ubiquitin Ligase MARCH6 Degrades Squalene Monooxygenase and Affects 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase and the Cholesterol Synthesis Pathway

Abstract

The mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis, is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect was not established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increases cell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation, we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytes and model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulator of flux through the cholesterol synthesis pathway.

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SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01140-13.

ACKNOWLEDGMENTS

We thank James Krycer, Sandhya Shrestha, and Vincenzo Sorrentino for developing various cell lines and Eric Reits for assistance with imaging. The microscopy images were taken at the core facility Cellular Imaging/LCAM-AMC.

A.J.B.'s lab is supported by grants from the National Health and Medical Research Council (1008081) and the National Heart Foundation of Australia (G11S5757), and N.Z.'s lab is supported by a Career Development Award from the Human Frontier Science Program Organization (HFSPO) and a VIDI grant (17.106.355) from the Netherlands Organization of Scientific Research (NWO).