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J. Biol. Chem., Vol. 280, Issue 27, 25811-25819, July 8, 2005

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Rapid Transbilayer Movement of Ceramides in Phospholipid Vesicles and in Human Erythrocytes^*

Iván López-Montero¶, Nicolas Rodriguez, Sophie Cribier, Antje Pohl, Marisela Vélez, and Philippe F. Devaux||

From the Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie 75005 Paris, France and the Instituto de Ciencia de los Materiales Nicolás Cabrera, C-XVI Universidad Autónoma de Madrid, E-28049 Cantoblanco Madrid, Spain

The transbilayer diffusion of unlabeled ceramides with different acyl chains (C₆-Cer, C₁₀-Cer, and C₁₆-Cer) was investigated in giant unilamellar vesicles (GUVs) and in human erythrocytes. Incorporation of a very small percentage of ceramides (0.1% of total lipids) to the external leaflet of egg phosphatidylcholine GUVs suffices to trigger a shape change from prolate to pear shape vesicle. By observing the reversibility of this shape change the transmembrane diffusion of lipids was inferred. We found a half-time for unlabeled ceramide flip-flop below 1 min at 37 °C. The rapid diffusion of ceramides in a phosphatidylcholine bilayer was confirmed by flip-flop experiments with a spin-labeled ceramide analogue incorporated into large unilamellar vesicles. Shape change experiments were also carried out with human erythrocytes to determine the trans-membrane diffusion of unlabeled ceramides into a biological membrane. Addition of exogenous ceramides to the external leaflet of human erythrocytes did not trigger echinocyte formation immediately as one would anticipate from an asymmetrical accumulation of new amphiphiles in the outer leaflet but only after 15 min of incubation at 20 °C in the presence of an excess of ceramide. We interpret these data as being indicative of a rapid ceramide equilibration between both erythrocyte leaflets as indicated also by electron spin resonance spectroscopy with a spin-labeled ceramide. The late appearance of echinocytes could reveal a progressive trapping of a fraction of the ceramide molecules in the outer erythrocytes leaflet. Thus, we cannot exclude the trapping of ceramides into plasma membrane domains.

Received for publication, October 25, 2004 , and in revised form, May 5, 2005.

^* This work was supported in part by grants from the CNRS (Unité Mixte de Recherche 7099), the Université Paris 7, and the Universidad Autónoma de Madrid. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of a fellowship from the Ministère de l'Education, de l'Enseignement Supérieur et de la Recherche Scientifique (Paris) and from the Fundación General de la Universidad Autonóma de Madrid.

^|| To whom correspondence should be addressed: Tel.: 33-1-58-41-5105; Fax: 33-1-58-41-5024; E-mail: Philippe.Devaux{at}ibpc.fr.

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