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

Roopa Kenoth, Rhoderick E. Brown, Ravi Kanth Kamlekar

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)237-256
Number of pages20
JournalMethods in molecular biology (Clifton, N.J.)
Volume1949
DOIs
StatePublished - Jan 1 2019

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Keywords

  • ACD11
  • Amphitropic protein
  • CPTP
  • FAPP2
  • Fluorescence
  • Förster resonance energy transfer
  • GLTP
  • GLTP-fold
  • Glycosphingolipid
  • HET-C2
  • Lipid vesicles
  • Membrane binding
  • Sphingolipid intermembrane transfer

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

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