Dissecting the contribution of sterol structure to membrane phase separation in living cells.

Abstract

The Bloch hypothesis states that the sterol biosynthesis pathway parallel's sterol structural evolution, where the product of each step should support optimized cellular and physiological functions when compared with its precursors. Here we explore this model in the yeast vacuole, which provides an in vivo system in which to explore the role of sterol structure to membrane phase separation. We compare the ability of sterol intermediates in the ergosterol biosynthesis pathway to support micron-sized phase separation by systematic genetic perturbations to the ergosterol biosynthesis pathway, driving the accumulation of different sterol intermediates in cells. Using fluorescence microscopy of stationary phase cells, we quantified changes in vacuole phase separation as a function of sterol species with the aid of the well-established vacuole associated protein marker Pho8-GFP. Interestingly, we observed a non-linear trend in the ability of sterol intermediates along the pathway to allow phase separation. Accumulation of early stage intermediates drove robust vacuole phase separation,while cells producing certain late stage intermediates exhibited little to no phase separation relative to the final product (ergosterol). Analogous experiments in model lipid membranes (giant unilamellar vesicles) corroborated our findings. These results suggest a more complex evolutionary trajectory for ergosterol biosynthesis, where minute structural differences in late stage intermediates do not predictably enhance membrane phase separation, contrary to the Bloch hypothesis. Thus, the structural features of ergosterol satisfy a fitness landscape in fungi where membrane phase separation is one of several functions lipid evolution selected for.