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Originally published In Press as doi:10.1194/jlr.D700023-JLR200 on September 28, 2007

Papers In Press, published online ahead of print January 1, 2008
J. Lipid Res., doi:10.1194/jlr.D700023-JLR200
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Journal of Lipid Research, Vol. 49, 251-262, January 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

Protein-sphingolipid interactions within cellular membranesboxs

Per Haberkant*, Oliver Schmitt*, F.-Xabier Contreras*, Christoph Thiele{dagger}, Kentaro Hanada§, Hein Sprong**, Constanze Reinhard{dagger}{dagger}, Felix T. Wieland1,* and Britta Brügger1,*

* Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany
{dagger} Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
§ Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
** Department of Membrane Enzymology, Bijvoet Center and Institute of Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands
{dagger}{dagger} Center for Human Genetics, Katholic University Leuven, 3000 Leuven, Belgium

boxs The online version of this article (available at http://www.jlr.org) contains Supplementary data in the form of 5 figures.

Published, JLR Papers in Press, September 28, 2007.

1 To whom correspondence should be addressed. e-mail: felix.wieland{at}bzh.uni-heidelberg.de (F.T.W.); britta.bruegger{at}bzh.uni-heidelberg.de (B.B.)

Each intracellular organelle critically depends on maintaining its specific lipid composition that in turn contributes to the biophysical properties of the membrane. With our knowledge increasing about the organization of membranes with defined microdomains of different lipid compositions, questions arise regarding the molecular mechanisms that underlie the targeting to/segregation from microdomains of a given protein. In addition to specific lipid-transmembrane segment interactions as a basis for partitioning, the presence in a given microdomain may alter the conformation of proteins and, thus, the activity and availability for regulatory modifications. However, for most proteins, the specific lipid environment of transmembrane segments as well as its relevance to protein function and overall membrane organization are largely unknown. To help fill this gap, we have synthesized a novel photoactive sphingolipid precursor that, together with a precursor for phosphoglycerolipids and with photo-cholesterol, was investigated in vivo with regard to specific protein transmembrane span-lipid interactions. As a proof of principle, we show specific labeling of the ceramide transporter with the sphingolipid probe and describe specific in vivo interactions of lipids with caveolin-1, phosphatidylinositol transfer protein β, and the mature form of nicastrin. This novel photolabile sphingolipid probe allows the detection of protein-sphingolipid interactions within the membrane bilayer of living cells.

Supplementary key words sphingosine • photoactivatable • ceramide transporter • phosphatidylinositol transfer protein • caveolin • nicastrin

Abbreviations: 10-ASA, 10-azi-stearic acid; Cer, ceramide; CERT, ceramide transporter; DRM, detergent-resistant membrane; FCS, fetal calf serum; MβCD, methyl-β-cyclodextrin; photoChol, photo-cholesterol; photoPC, photo-phosphatidylcholine; photoSph, photo-sphingosine; PI-TP, phosphatidylinositol transfer protein; PVDF, polyvinylidene difluoride; SM, sphingomyelin; UV, ultraviolet


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