Sterol analysis of MARCHF6-deficient cells reveals a new pinch point in the cholesterol biosynthesis pathway

• The E3 ubiquitin ligase MARCHF6 was previously found to degrade six enzymes within the cholesterol biosynthesis pathway, including the canonical substrate, SQLE (squalene monooxygenase). • MARCHF6 knockout HEK293T cells were generated using CRISPR to compare sterol intermediate profiles with wild-type cells using radio-TLC and GC/MS methods. • MARCHF6 knockout cells have similar total cholesterol levels but exhibit major differences in flux of sterol intermediates throughout the pathway. • Flux through SQLE is especially enhanced in MARCHF6 knockout cells, increasing production of the oxysterol 24,25EC via the shunt pathway. • Notably, our work also identifies SC5D as a previously unrecognized pinch point in the pathway.