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In Vitro Measurement of Sphingolipid Intermembrane Transport Illustrated by GLTP Superfamily Members

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Intracellular Lipid Transport

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1949))

Abstract

Herein, we describe methodological approaches for measuring in vitro transfer of sphingolipids (SLs) between membranes. The approaches rely on direct tracking of the lipid. Typically, direct tracking involves lipid labeling via attachment of fluorophores or introduction of radioactivity. Members of the GlycoLipid Transfer Protein (GLTP) superfamily are used to illustrate two broadly applicable methods for direct lipid tracking. One method relies on Förster resonance energy transfer (FRET) that enables continuous assessment of fluorophore-labeled SL transfer in real time between lipid donor and acceptor vesicles. The second method relies on tracking of radiolabeled SL transfer by separation of lipid donor and acceptor vesicles at discrete time points. The assays are readily adjustable for assessing lipid transfer (1) between various model membrane assemblies (vesicles, micelles, bicelles, nanodiscs), (2) involving other lipid types by other lipid transfer proteins, (3) with protein preparations that are either crudely or highly purified, and (4) that is spontaneous and occurs in the absence of protein.

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Acknowledgments

We are grateful to many individuals who contributed to the development of the experimental approaches used routinely in the REB lab for many years and detailed here. They include J.G. Molotkovsky, H.M. Pike, X Zhai, I.A. Boldyrev, Y-G Gao, and P. Mattjus. We also are grateful for support from Dept. of Science and Technology, Science and Engineering Research Board (SERB), Govt. of India to RKK (YSS/2014/000021) and to RK (YSS/2015/000783) as well as support by NIH/NIGMS-GM45928, NIH/NCI-CA121493, and NIH/NHLBI-HL125353 (to REB) and the Hormel Foundation. We thank VIT for research facilities and infrastructure.

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Correspondence to Rhoderick E. Brown or Ravi Kanth Kamlekar .

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Kenoth, R., Brown, R.E., Kamlekar, R.K. (2019). In Vitro Measurement of Sphingolipid Intermembrane Transport Illustrated by GLTP Superfamily Members. In: Drin, G. (eds) Intracellular Lipid Transport. Methods in Molecular Biology, vol 1949. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9136-5_17

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