title: The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: purification, characterization, and the effect of lipids on its activity.
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 was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze 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 that is critical for the activity of the transporter.
authors.affiliation: Institute of Biology, Humboldt University of Berlin, Invalidenstrasse 42, 10115 Berlin, Germany.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Marek
authors.forename: Sigrid
authors.initials: S
authors.lastname: Milles
authors.forename: Gabriele
authors.initials: G
authors.lastname: Schreiber
authors.forename: David L
authors.initials: DL
authors.lastname: Daleke
authors.forename: Gunnar
authors.initials: G
authors.lastname: Dittmar
authors.forename: Andreas
authors.initials: A
authors.lastname: Herrmann
authors.forename: Peter
authors.initials: P
authors.lastname: Müller
authors.forename: Thomas Günther
authors.initials: TG
authors.lastname: Pomorski
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006868:Hydrolysis; D020128:Inhibitory Concentration 50; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods; D009711:Nucleotides / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; C466994:AUS1 protein, S cerevisiae; D008055:Lipids; D008081:Liposomes; D009711:Nucleotides; D010718:Phosphatidylserines; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D008715:Methionine; D000251:Adenosine Triphosphatases; D008239:Lysine