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

Lukasz M. Solanko, David P. Sullivan, Yves Y. Sere, Maria Szomek, Anita Lunding, Katarzyna A. Solanko, Azra Pizovic, Lyubomir D. Stanchev, Thomas Günther Pomorski*, Anant K. Menon, Daniel Wüstner

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Resumé

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.

OriginalsprogEngelsk
TidsskriftTraffic
Vol/bind19
Udgave nummer3
Sider (fra-til)198-214
ISSN1398-9219
DOI
StatusUdgivet - 2018

Fingeraftryk

Ergosterol
Cell membranes
Yeast
Cell Membrane
Sterols
Phospholipids
Quenching
Fluorescence
Trinitrobenzenes
Lipids
Sphingolipids
Lipid bilayers
Sulfonic Acids
Spectrophotometry
Lipid Bilayers
Eukaryotic Cells
Membrane Lipids
Phosphatidylcholines
Fluorescence Microscopy
Glass

Citer dette

Solanko, Lukasz M. ; Sullivan, David P. ; Sere, Yves Y. ; Szomek, Maria ; Lunding, Anita ; Solanko, Katarzyna A. ; Pizovic, Azra ; Stanchev, Lyubomir D. ; Pomorski, Thomas Günther ; Menon, Anant K. ; Wüstner, Daniel. / Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane. I: Traffic. 2018 ; Bind 19, Nr. 3. s. 198-214.
@article{d478f65b5f4e4d08873358a925d69a6a,
title = "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.",
keywords = "asymmetry, collisional quenching, dehydroergosterol, ergosterol, fluorescence, heme, plasma membrane, TNBS, yeast",
author = "Solanko, {Lukasz M.} and Sullivan, {David P.} and Sere, {Yves Y.} and Maria Szomek and Anita Lunding and Solanko, {Katarzyna A.} and Azra Pizovic and Stanchev, {Lyubomir D.} and Pomorski, {Thomas G{\"u}nther} and Menon, {Anant K.} and Daniel W{\"u}stner",
year = "2018",
doi = "10.1111/tra.12545",
language = "English",
volume = "19",
pages = "198--214",
journal = "Traffic",
issn = "1398-9219",
publisher = "Wiley-Blackwell",
number = "3",

}

Solanko, LM, Sullivan, DP, Sere, YY, Szomek, M, Lunding, A, Solanko, KA, Pizovic, A, Stanchev, LD, Pomorski, TG, Menon, AK & Wüstner, D 2018, 'Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane', Traffic, bind 19, nr. 3, s. 198-214. https://doi.org/10.1111/tra.12545

Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane. / Solanko, Lukasz M.; Sullivan, David P.; Sere, Yves Y.; Szomek, Maria; Lunding, Anita; Solanko, Katarzyna A.; Pizovic, Azra; Stanchev, Lyubomir D.; Pomorski, Thomas Günther; Menon, Anant K.; Wüstner, Daniel.

I: Traffic, Bind 19, Nr. 3, 2018, s. 198-214.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane

AU - Solanko, Lukasz M.

AU - Sullivan, David P.

AU - Sere, Yves Y.

AU - Szomek, Maria

AU - Lunding, Anita

AU - Solanko, Katarzyna A.

AU - Pizovic, Azra

AU - Stanchev, Lyubomir D.

AU - Pomorski, Thomas Günther

AU - Menon, Anant K.

AU - Wüstner, Daniel

PY - 2018

Y1 - 2018

N2 - 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.

AB - 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.

KW - asymmetry

KW - collisional quenching

KW - dehydroergosterol

KW - ergosterol

KW - fluorescence

KW - heme

KW - plasma membrane

KW - TNBS

KW - yeast

U2 - 10.1111/tra.12545

DO - 10.1111/tra.12545

M3 - Journal article

C2 - 29282820

AN - SCOPUS:85042465022

VL - 19

SP - 198

EP - 214

JO - Traffic

JF - Traffic

SN - 1398-9219

IS - 3

ER -