The role of ABC proteins Aus1p and Pdr11p in the uptake of external sterols in yeast: dehydroergosterol fluorescence study.
Uptake of external sterols in the yeast Saccharomyces cerevisiae is a multistep process limited to anaerobiosis or heme deficiency. It includes crossing the cell wall, insertion of sterol molecules into plasma membrane and their internalization and integration into intracellular membranes. We applied the fluorescent ergosterol analog dehydroergosterol (DHE) to monitor the initial steps of sterol uptake by three independent approaches: fluorescence spectroscopy, fluorescence microscopy and sterol quantification by HPLC. Using specific fluorescence characteristics of DHE we showed that the entry of sterol molecules into plasma membrane is not spontaneous but requires assistance of two ABC (ATP-binding cassette) pumps--Aus1p or Pdr11p. DHE taken up by uptake-competent hem1ΔAUS1PDR11 cells could be directly visualized by UV-sensitive wide field fluorescence microscopy. HPLC analysis of sterols revealed significant amounts of exogenous ergosterol and DHE (but not cholesterol) associated with uptake-deficient hem1Δaus1Δpdr11Δ cells. Fluorescent sterol associated with these cells did not show the characteristic emission spectrum of membrane-integrated DHE. The amount of cell-associated DHE was significantly reduced after enzymatic removal of the cell wall. Our results demonstrate that the yeast cell wall is actively involved in binding and uptake of ergosterol-like sterols.
Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Ivanka pri Dunaji 90210, Slovak Republic.
Peter
P
Kohut
Daniel
D
Wüstner
Lucia
L
Hronska
Karl
K
Kuchler
Ivan
I
Hapala
Martin
M
Valachovic
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000502:physiology*; D001692:Biological Transport; D002851:Chromatography, High Pressure Liquid; D004875:Ergosterol / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*; D013261:Sterols / Q000032:analysis / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; C466994:AUS1 protein, S cerevisiae; D005456:Fluorescent Dyes; C466995:PDR11 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013261:Sterols; C010905:dehydroergosterol; D004875:Ergosterol