Specific proteins are required to translocate phosphatidylcholine bidirectionally across the endoplasmic reticulum.
BACKGROUND
A long-standing problem in understanding the mechanism by which the phospholipid bilayer of biological membranes is assembled concerns how phospholipids flip back and forth between the two leaflets of the bilayer. This question is important because phospholipid biosynthetic enzymes typically face the cytosol and deposit newly synthesized phospholipids in the cytosolic leaflet of biogenic membranes such as the endoplasmic reticulum (ER). These lipids must be transported across the bilayer to populate the exoplasmic leaflet for membrane growth. Transport does not occur spontaneously and it is presumed that specific membrane proteins, flippases, are responsible for phospholipid flip-flop. No biogenic membrane flippases have been identified and there is controversy as to whether proteins are involved at all, whether any membrane protein is sufficient, or whether non-bilayer arrangements of lipids support flip-flop.


RESULTS
To test the hypothesis that specific proteins facilitate phospholipid flip-flop in the ER, we reconstituted transport-active proteoliposomes from detergent-solubilized ER vesicles under conditions in which protein-free liposomes containing ER lipids were inactive. Transport was measured using a synthetic, water-soluble phosphatidylcholine and was found to be sensitive to proteolysis and associated with proteins or protein-containing complexes that sedimented operationally at 3.8S. Chromatographic analyses indicated the feasibility of identifying the transporter(s) by protein purification approaches, and raised the possibility that at least two different proteins are able to facilitate transport. Calculations based on a simple reconstitution scenario suggested that the transporters represent approximately 0.2% of ER membrane proteins.


CONCLUSIONS
Our results clearly show that specific proteins are required to translocate a phosphatidylcholine analogue across the ER membrane. These proteins are likely to be the flippases, which are required to translocate natural phosphatidylcholine and other phospholipids across the ER membrane. The methodology that we describe paves the way for identification of a flippase.
Menon
A K
AK
Department of Biochemistry, University of Wisconsin-Madison, Madison, 53706-1569, USA. menon@biochem.wisc.edu.
Watkins WE3rd
Hrafnsdóttir
S
S
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D019286:Endopeptidase K / Q000494:pharmacology; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000378:metabolism; D008954:Models, Biological; D017830:Octoxynol / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D013997:Time Factors
D002352:Carrier Proteins; D003902:Detergents; D008081:Liposomes; D008565:Membrane Proteins; D010713:Phosphatidylcholines; D049294:Phospholipid Transfer Proteins; D011510:Proteolipids; C020485:proteoliposomes; D017830:Octoxynol; D019286:Endopeptidase K