Tritium suicide selection identifies proteins involved in the uptake and intracellular transport of sterols in Saccharomyces cerevisiae.
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.
Sullivan
David P
DP
Department of Biochemistry, Weill Cornell Medical College, 1300 York Ave., New York, NY 10065, USA.
Georgiev
Alexander
A
Menon
Anant K
AK
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D016296:Mutagenesis; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013261:Sterols / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014316:Tritium / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; C466994:AUS1 protein, S cerevisiae; D002352:Carrier Proteins; C538989:DET1 protein, S cerevisiae; D004268:DNA-Binding Proteins; C112006:MOT3 protein, S cerevisiae; C085480:PDR1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013261:Sterols; D015534:Trans-Activators; D014157:Transcription Factors; D014316:Tritium