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The yeast plasma membrane ABC transporter Aus1: Purification, characterization and effect of lipids on its activity

Item Type:Article
Title:The yeast plasma membrane ABC transporter Aus1: Purification, characterization and effect of lipids on its activity
Creators Name:Marek, M. and Milles, S. and Schreiber, G. and Daleke, D.L. and Dittmar, G. and Herrmann, A. and Mueller, P. and Pomorski, T.G.
Abstract:The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure has been developed to characterize the Aus1 transporter. The detergent solubilised protein was able to bind and hydrolyse ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS which is crucial for the activity of the transporter.
Keywords:ABC Transporter, Fluorescence, Phosphatidylserine, Sterol, Yeast, ATP Hydrolysis, Proteoliposomes
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:286
Number:24
Page Range:21835-21843
Date:17 June 2011
Official Publication:https://doi.org/10.1074/jbc.M111.244525
PubMed:View item in PubMed

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