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. 2009 Feb;8(2):161-9.
doi: 10.1128/EC.00135-08. Epub 2008 Dec 5.

Tritium suicide selection identifies proteins involved in the uptake and intracellular transport of sterols in Saccharomyces cerevisiae

David P Sullivan  1 , Alexander Georgiev, Anant K Menon
Affiliations

Tritium suicide selection identifies proteins involved in the uptake and intracellular transport of sterols in Saccharomyces cerevisiae

David P Sullivan et al. Eukaryot Cell. 2009 Feb.

Abstract

Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [(3)H]cholesterol in the form of [(3)H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [(3)H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [(3)H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Delta cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport.

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Figures

FIG. 1.
FIG. 1.
[3H]cholesterol suicide selection. (A) Schematic illustration showing the uptake, PM-ER transport, and accumulation of cholesterol in the form of cholesteryl ester in a Upc2-1 cell. (B) Killing curve. Upc2-1 cells were mutagenized with a transposon library. After outgrowth, 8 × 106 cells were either loaded with [3H]cholesterol or mock treated with nonlabeled cholesterol. Aliquots of the cells were frozen and stored at −80°C. At the indicated times, aliquots were removed from the freezer, thawed, and plated to determine the number of surviving colonies. The graph shows the percent survival of 3H-loaded and mock-treated cells after storage at −80°C. (C) Colonies that survived the suicide selection were gridded, replica plated onto nitrocellulose paper, and overlaid on agar containing [14C]cholesterol. After incubation, colony-associated radioactivity was visualized with a phosphorimager. Open arrowheads indicate examples of colonies with decreased uptake that were picked for further study.
FIG. 2.
FIG. 2.
Quantitative assay of sterol incorporation by a subset of suicide selection survivors. (A) Logarithmically growing cultures of isolates from the suicide selection were incubated in YPD with 1 μM cholesterol (containing 10 μCi [3H]cholesterol) in 0.5% (wt/vol) ethanol-0.5% (wt/vol) Tween 80 for 60 min at 37°C. Cells were recovered, washed, and lysed. The incorporation of [3H]cholesterol was determined by scintillation counting and extrapolated to the amount of bulk cholesterol uptake in the culture. The graph shows the averages and variances for two independent experiments. (B) Lysed cells from panel A were taken for lipid extraction; free cholesterol and cholesteryl esters in the extract were resolved by TLC and quantitated as described in Materials and Methods.
FIG. 3.
FIG. 3.
Effect of Mot3 on expression of Aus1 and Pdr11. Logarithmically growing cultures of the Upc2-1 strain, a mutant with a transposon insertion in the TVP18 locus, and Upc2-1 cells transformed with a single-copy plasmid containing MOT3 (pMOT3) were preincubated at 37°C for 45 min. Cells were recovered, and RNAs were extracted and transcribed into DNA as described in Materials and Methods. After cDNA synthesis, the levels of mRNA corresponding to MOT3, AUS1, and PDR11 were determined using QPCR. Data (normalized to results for the Upc2-1 strain) show the averages and variances for two separate experiments.
FIG. 4.
FIG. 4.
Aus1-GFP and Pdr11-GFP localization. Mutants from the suicide selection were transformed with single-copy vectors bearing either AUS1-GFP or PDR11-GFP under the control of endogenous promoters. The strains were grown to mid-logarithmic phase in synthetic media lacking the appropriate nutrient and visualized using wide-field fluorescence microscopy. Images were generated under the same settings and processed identically. Representative cells are shown. Bar = 10 μm.
FIG. 5.
FIG. 5.
Sterol transport in det1Δ cells. Sterol transport in BY4741 (parent) and det1Δ cells was measured by using MβCD to monitor the arrival of metabolically labeled ergosterol at the PM in a pulse-chase experiment as described in Materials and Methods. The RSR of [3H]ergosterol in the MβCD extract was quantitated and graphed versus chase time. The lines represent mono-exponential fits of data averaged from at least two independent experiments. Error bars represent standard deviations.
FIG. 6.
FIG. 6.
Lipid droplets and subcellular distribution of ergosterol in det1Δ cells. (A) Lipid droplets in BY4741 (parent) and det1Δ cells were visualized by fluorescence microscopy after Nile red staining. Bar = 10 μm. (B) Fractionation of a BY4741 cell homogenate on a discontinuous RenoCal gradient as described in Materials and Methods. Fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting, using antibodies against the PM (Gas1), vacuole (Vph1), ER (sec61), and Golgi complex/endosome (Pep12). Fractions 1 to 8, corresponding to intracellular membranes, and fractions 9 to 14, corresponding to the PM, were pooled and taken for lipid analysis. (C) Percentages of total ergosterol recovered in PM pools isolated from BY4741 and det1Δ cells.
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