Flip-flop of fluorescently labeled phospholipids in proteoliposomes reconstituted with Saccharomyces cerevisiae microsomal proteins.
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-7-nitrobenz-2-oxa-1,3-diazol-4-yl (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 analogues 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 approximately 4S protein fraction enriched in flippase activity as well as of other fractions where flippase activity was depleted or undetectable. We estimate that flippase activity is due to a protein(s) representing approximately 2% (wt/wt) 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.
Vehring
Stefanie
S
Institute of Biology/Biophysics, Humboldt University of Berlin, Invalidenstr. 42, 10115 Berlin, Germany.
Pakkiri
Leroy
L
Schröer
Adrien
A
Alder-Baerens
Nele
N
Herrmann
Andreas
A
Menon
Anant K
AK
Pomorski
Thomas
T
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D007425:Intracellular Membranes / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D026901:Membrane Transport Proteins; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D017830:Octoxynol / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D049294:Phospholipid Transfer Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry; D021381:Protein Transport; D011510:Proteolipids / Q000737:chemistry*; D000069816:SEC Translocation Channels; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C078632:1-acyl-2-(12-((7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl)phosphatidylcholine; D008051:Lipid Bilayers; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010713:Phosphatidylcholines; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D011510:Proteolipids; D000069816:SEC Translocation Channels; C499383:SEC61 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C020485:proteoliposomes; D017830:Octoxynol; D009327:4-Chloro-7-nitrobenzofurazan