Dissection of ergosterol metabolism reveals a pathway optimized for membrane phase separation

Sterols are among the most abundant lipids in eukaryotic cells, yet are synthesized through notoriously long metabolic pathways. It has been proposed that the molecular evolution of such pathways must have required each step to increase the capacity of its product to condense and order phospholipids. Here we carry out a systematic analysis of the ergosterol pathway that leverages the yeast vacuole's capacity to phase-separate as a predictive biophysical readout for each intermediate. In the post-synthetic steps specific to ergosterol biosynthesis, we find that successive modifications act to oscillate ordering capacity, settling on a level that supports phase separation while retaining fluidity of the resulting domains. Simulations carried out with each intermediate showed how conformers in the sterol's alkyl tail are capable of modulating long-range ordering of phospholipids, which could underlie changes in phase behavior. Our results indicate that the complexity of sterol metabolism could have resulted from the need to balance lipid interactions required for membrane organization.