Measuring translocation of fluorescent lipid derivatives across yeast Golgi membranes.
Phospholipid asymmetry is a fundamental feature of the plasma membrane of most eukaryotic cells and its regulation is linked to diverse physiological processes such as apoptosis and blood clotting [P. Williamson, R.A. Schlegel, Biochim. Biophys. Acta 1585 (2002) 53-63; R.F. Zwaal, A.J. Schroit, Blood 89 (1997) 1121-1132]. In addition, the phospholipid translocases (flippases) that are thought to establish asymmetry are also implicated in vesicle-mediated protein transport throughout the secretory and endocytic pathways [T.R. Graham, Trends Cell Biol. 14 (2004) 670-677]. However, the biochemical properties of phospholipid translocases in membranes of the Golgi complex and endosomes have received much less attention than translocases in the plasma membrane. We describe here a method for purifying yeast Golgi membranes and assaying an ATP-dependent phospholipid translocase activity in these membranes using fluorescent lipid analogues. This assay detects ATP-dependent translocation of labeled phosphatidylserine across late Golgi membranes, which requires the activity of a P-type ATPase called Drs2p [P. Natarajan, J. Wang, Z. Hua, T.R. Graham, Proc. Natl. Acad. Sci. USA 101 (2004) 10614-10619].
Natarajan
Paramasivam
P
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA.
Graham
Todd R
TR
D000255:Adenosine Triphosphate / Q000378:metabolism; D001393:Azoles / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D001692:Biological Transport; D005456:Fluorescent Dyes / Q000032:analysis; D006056:Golgi Apparatus / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D050356:Lipid Metabolism; D008055:Lipids / Q000032:analysis*; D009578:Nitrobenzenes / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000032:analysis* / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis / Q000378:metabolism; D015003:Yeasts / Q000378:metabolism
C510797:7-nitrobenz-2-oxa-1,3-diazol-4-yl; D001393:Azoles; D005456:Fluorescent Dyes; D008055:Lipids; D009578:Nitrobenzenes; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D000255:Adenosine Triphosphate