Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane

Abstract
Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol‐auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using spectrophotometry and fluorescence microscopy we found that <20% of DHE fluorescence was quenched when the DHE‐containing cells were exposed to membrane‐impermeant collisional quenchers (spin‐labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass‐bead lysis or repeated freeze‐thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Likewise, an increase in the quenchable pool of DHE w...

Source: Traffic - December 28, 2017 Category: Research Authors: Lukasz M. Solanko, David P. Sullivan, Yves Y. Sere, Maria Szomek, Anita Lunding, Katarzyna A. Solanko, Azra Pizovic, Lyubomir D. Stanchev, Thomas G ünther Pomorski, Anant K. Menon, Daniel Wüstner Tags: ORIGINAL ARTICLE Source Type: research