Journal
CURRENT BIOLOGY
Volume 10, Issue 5, Pages 241-252Publisher
CELL PRESS
DOI: 10.1016/S0960-9822(00)00356-0
Keywords
-
Categories
Funding
- NIGMS NIH HHS [GM55427] Funding Source: Medline
Ask authors/readers for more resources
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 flipflop 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 similar to 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available