Eukaryotic Cell doi:10.1128/EC.00198-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Flip-flop of fluorescently labeled phospholipids in proteoliposomes reconstituted with yeast microsomal proteins
Stefanie Vehring,
Leroy Pakkiri,
Adrien Schröer,
Nele Alder-Baerens,
Andreas Herrmann,
Anant K. Menon*,
and
Thomas Pomorski*
Institute of Biology/Biophysics, Humboldt University of Berlin, Invalidenstr. 42, 10115 Berlin, Germany; and Department of Biochemistry, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10021, USA
* To whom correspondence should be addressed. Email:
akm2003{at}med.cornell.edu. thomas.pomorski{at}rz.hu-berlin.de.
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Abstract |
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A phospholipid flippase activity from the endoplasmic reticulum (ER) of the model organism Saccharomyces cerevisiae has been characterized and functionally reconstituted into proteoliposomes. Analysis of the transbilayer movement of acyl-NBD-labeled phosphatidylcholine in yeast microsomes using a fluorescence stopped-flow back exchange assay revealed a rapid, ATP-independent flip-flop (half-time <2 min). Proteoliposomes prepared from a Triton X-100 extract of yeast microsomal membranes were also capable of flipping NBD-labeled phospholipid analogs rapidly in an ATP-independent fashion. Flippase activity was sensitive to the protein modification reagents N-ethylmaleimide and diethylpyrocarbonate. Resolution of the Triton X-100 extract by velocity gradient centrifugation resulted in the identification of a 
4S protein fraction enriched in flippase activity as well as other fractions where flippase activity was depleted or undetectable. We estimate that flippase activity is due to a protein(s) representing 2% (w/w) of proteins in the Triton X-100 extract. These results indicate that specific proteins are required to facilitate ATP-independent phospholipid flip-flop in the ER and that their identification is feasible. The architecture of the ER protein translocon suggests that it could account for the flippase activity in the ER. We tested this hypothesis using microsomes prepared from a temperature-sensitive yeast mutant in which the major translocon component, Sec61p, was quantitatively depleted. We found that the protein translocon is not required for transbilayer movement of phospholipids across the ER. Our work defines yeast as a promising model system for future attempts to identify the ER phospholipid flippase and to test and purify candidate flippases.
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