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

doi:10.1074/jbc.271.12.6651

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

From the (1)Laboratoire de Biophysique Cellulaire, URA 526 Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris and
(2)Université Denis Diderot, 2 place Jussieu, 75251 Paris Cedex 05, France

^§ Recipient of a fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche. To whom correspondence should be addressed.

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 P-NMR. Several conditions were found under which the P 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.

Footnotes

↵* This research was supported by a European Community Biotechnology contract (BIO2-CT93-0348) and by grants from the Centre National de la Recherche Scientifique (URA 526) and the Université Paris 7-Denis Diderot. The NMR spectrometer used in this study was purchased with funds from the Centre National de la Recherche Scientifique (Equipements mi-lourds et ATIPE No. 1), the Université Paris 7-Denis Diderot, the Institut National pour la Santé et la Recherche Médicale, and the Association pour la Recherche sur le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SL

spin-labeled

BSA

bovine serum albumin

diCPC

dibutyroylphosphatidylcholine

ER

endoplasmic reticulum

LPC

lysophosphatidylcholine

PC

phosphatidylcholine

PE

phosphatidylethanolamine

PS

phosphatidylserine

SM

sphingomyelin.
- Received June 7, 1995.
- Revision received January 9, 1996.