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 fluorescence spectrophotometry and 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. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer.

Transverse distribution of plasma membrane bilayer cholesterol: Picking sides

“…In a recent study, the natural ergosterol in an auxotroph of Saccharomyces cerevisiae was completely replaced by dehydroergosterol (DHE), enabling the surface accessibility of the sterol to be determined with impermeant fluorescence quenchers, as described above. 75 An exhaustive study suggested that~70% of cellular DHE was located in the plasma membrane and~80% of that resided the inner leaflet thereof, in agreement with the aforementioned studies using the DHE quenching approach. This analysis took into account both the incomplete quenching of accessible DHE and the (inaccessible) intracellular pool which would otherwise be scored with the cytoplasmic leaflet.…”

“…However, this appears not to be the case for intact yeast cells (Müller et al, personal communication). Also, 4‐SLPC gave results comparable to TNBS, seeming to validate its use . Like cholesterol itself, DHE can move rapidly between the plasma membrane and intracellular compartments .…”

“…Various control studies have shown that DHE and CTL are, in many respects, benign and faithful mimics of and substitutes for cholesterol both physically and physiologically . (Indeed, DHE is a natural constituent of yeast plasma membranes.)…”

“…Various control studies have shown that DHE and CTL are, in many respects, benign and faithful mimics of and substitutes for cholesterol both physically and physiologically. [74][75][76]83,[85][86][87] (Indeed, DHE is a natural constituent of yeast plasma membranes.) Nevertheless, these are not ideal probes for technical reasons: they have a low-quantum yield, photobleach rapidly and require microscope optics in the near ultraviolet.…”

Avidity‐driven polarity establishment via multivalent lipid– GTP ase module interactions

“…In budding yeast, Bem1 is proposed to play a role in this feedback (Bose et al, 2001;Goryachev & Pokhilko, 2008;Witte et al, 2017); however, the mechanisms that localize Bem1 to the plasma membrane to trigger the positive feedback have been enigmatic. Interestingly, ergosterol levels have been reported to be non-homogeneous in the yeast plasma membrane, where it was observed to cluster, although another study reported a more homogeneous distribution (Grossmann et al, 2007;Solanko et al, 2018). Moreover, our solid-state NMR spectroscopy experiments suggest that the interaction between Bem1 and anionic lipids is reciprocal in the sense that Bem1 influences the ordering of lipid acyl chains, rigidifying the local membrane environment where ergosterol levels are low, while fluidizing it where ergosterol levels are high.…”