Ultrafast Glycerophospholipid-selective Transbilayer Motion Mediated by a Protein in the Endoplasmic Reticulum Membrane

Abstract

A relatively rapid transbilayer motion of phospholipids in the microsomal membrane seems to be required due to their asymmetric synthesis in the cytoplasmic leaflet. Marked discrepancies exist with regard to the rate and specificity of this flip-flop process. To reinvestigate this problem, we have used both spin-labeled and radioactively labeled long chain phospholipids with a new fast translocation assay. Identical results were obtained with both types of probes. Transbilayer motion of glycerophospholipids was found to be much more rapid than previously reported (half-time less than 25 s) and to occur identically for phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. Such transport is nonvectorial and leads to a symmetric transbilayer distribution of phospholipids. In contrast, transverse diffusion of sphingomyelin was 1 order of magnitude slower. Phospholipid flip-flop appears to occur by a protein-mediated transport process displaying saturable and competitive behavior. Proteolysis, chemical modification, and competition experiments suggest that this transport process may be related to that previously described in the endoplasmic reticulum for short-chain phosphatidylcholine (Bishop, W. R., and Bell, R. M. (1985) Cell 42, 51-60). The relationship between phospholipid flip-flop and nonbilayer structures occurring in the endoplasmic reticulum was also investigated by 31P-NMR. Several conditions were found under which the 31P isotropic NMR signal previously attributed to nonbilayer structures is decreased or abolished, whereas transbilayer diffusion is unaffected, suggesting that the flip-flop process is independent of such structures. It is concluded that flip-flop in the endoplasmic reticulum is mediated by a bidirectional protein transporter with a high efficiency for glycerophospholipids and a low efficiency for sphingomyelin. In vivo, the activity of this transporter would be able to redistribute all changes in phospholipid composition due to biosynthetic processes between the two leaflets of the endoplasmic reticulum membranes within a time scale of seconds.

Ethanol specifically alters the synthesis, acylation and transbilayer movement of aminophospholipids in rat-liver microsomes

“…In a similar way, a rapid flip-flop has been reported in microsomal membranes at half-times of seconds to minutes (Buton et al, 1996) in a process that has proved to be ATPindependent and protein-mediated. It has been suggested that this flipping machinery could translocate most, if not all, of the phospholipid classes across the bilayer non-vectorially (Kol et al, 2002).…”

“…The transverse distribution of phospholipids in the ER, on the other hand, is still a matter of debate after conflicting observations (Devaux, 1993). Nevertheless, recent publications suggest that the distribution of most phospholipid classes in the ER is largely symmetrical (Buton et al, 1996;Kol et al, 2002). Thus it is important to view the biosynthesis of PS and PE in the biogenic membrane, ER, as being an asymmetric process that results in the insertion of new lipids into the cytoplasmic leaflet.…”

Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum

“…4 and 2A, there is a general trend that the membrane-insertion efficiency is higher when the alkyl side chain is in the lumenal half of the H segment (i.e., the asymmetry in the ΔG app curves correlates with the membrane orientation of the H segment), suggesting that it reflects an asymmetry in either the ER membrane or the Sec61 translocon. Although data are scarce, the ER is thought not to have a strong asymmetry in lipid composition between the two leaflets (17,18). Therefore, it is more likely that the origin of the asymmetry is to be sought in the translocon.…”

Sphingolipid transport in eukaryotic cells

“…This could be mediated by the activation of the scramblase, but, alternatively, SM could £ip across the membrane in a more proximal part of the secretory pathway. Indeed, evidence has been provided for a bidirectional translocation of phospholipids across the ER membrane, whereby SM translocation was slower than that of the glycerophospholipids [245,246]. Whether this rapid £ip-£op is due to a`£ippase' or to non-bilayer structures in the ER membrane has been debated for years [65].…”