Role of membrane contact sites in PM ‐ ER sterol transport (606.7)

Abstract

Intracellular trafficking of sterols occurs largely by non‐vesicular processes that are poorly understood. Cytoplasmic sterol transport proteins (STPs) are presumed to ferry sterols between membranes and this may be enhanced in regions where the participating membranes are closely apposed (membrane contact sites or MCSs). The major sterol trafficking pathway in the yeast Saccharomyces cerevisiae involves direct transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). Yeast STPs have not been identified, although the cytoplasmic protein Kes1/Osh4 has been implicated in the phosphoinositide‐driven cycling of ergosterol between the ER and TGN. We considered whether MCSs between the ER and PM might play a role in sterol transport between the two organelles. We tested this hypothesis by analyzing sterol traffic in Saccharomyces cerevisiae cells that were largely devoid of ER ‐ PM contact sites (∆tether cells). We used fluorescence microscopy to track the retrograde movement of exogenously supplied dehydroergosterol (DHE) from the PM to the ER and lipid droplets, and high performance liquid chromatography to quantify, in parallel, the transport‐coupled formation of DHE esters. We found that sterol transport from PM to ER is unaffected in ∆tether cells. We are currently generating strains based on ∆tether cells in which all residual ER‐PM MCSs are eliminated, and also testing whether vesicular pathways might play a redundant role in the absence of MCSs.