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Eukaryotic Cell, February 2009, p. 161-169, Vol. 8, No. 2
1535-9778/09/$08.00+0     doi:10.1128/EC.00135-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae{triangledown}

David P. Sullivan, Alexander Georgiev, and Anant K. Menon*

Department of Biochemistry, Weill Cornell Medical College, 1300 York Ave., New York, New York 10065

Received 15 April 2008/ Accepted 24 November 2008

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 [3H]cholesterol in the form of [3H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [3H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [3H]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 det1{Delta} cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport.

* Corresponding author. Mailing address: Department of Biochemistry, Weill Cornell Medical College, 1300 York Ave., New York, NY 10065. Phone: (212) 746-5941. Fax: (212) 746-8875. E-mail: akm2003{at}med.cornell.edu

{triangledown} Published ahead of print on 5 December 2008.

Eukaryotic Cell, February 2009, p. 161-169, Vol. 8, No. 2
1535-9778/09/$08.00+0     doi:10.1128/EC.00135-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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