Abstract
Mammalian cells have evolved proteins that can extract single lipids from membranes and sequester them in their hydrophobic cavity. These proteins, collectively known as lipid transport proteins, play essential roles in many aspects of lipid metabolism, lipid signaling, and membrane traffic. Phosphatidylinositol transfer proteins (α and β) are lipid transport proteins that specifically bind phosphatidylinositol or phosphatidylcholine in their hydrophobic cavity and facilitate their transfer from one membrane compartment to another. In addition, PITPβ can facilitate sphingomyelin transfer. This chapter describes methods to monitor the transfer and binding activity of PITPs using a variety of assays, including an assay that uses microsomes as a donor and liposomes as an acceptor of PtdIns. For phosphatidylcholine and sphingomyelin transfer, liposomes are used as a donor compartment and mitochondria as an acceptor compartment. A separate method describes the use of permeabilized cells as a source of donor lipids and liposomes as an acceptor membrane. In addition to lipid transfer, methods to identify the lipids that occupy the hydrophobic cavity of PITPs are discussed.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsAbbreviations
- PtdIns::
-
Phosphatidylinositol;
- PtdCho::
-
Phosphatidylcholine;
- SM::
-
Sphingomyelin;
- PITP::
-
Phosphatidylinositol transfer proteins;
- LTP::
-
Lipid transfer proteins;
- PtdOH::
-
Phosphatidic acid;
- PtdEth::
-
Phosphatidylethanolamine.
References
Helmkamp GM, Jr, Harvey MS, Wirtz KWA, van Deenen LLM. Phospholipid exchange between membranes. Purification of bovine brain proteins that preferentially catalyze the transfer of phosphatidylinositol. J Biol Chem 1974;249:6382–9.
Cockcroft S. Phosphatidylinositol transfer proteins: A requirement in signal transduction and vesicle traffic. BioEssays 1998;20:423–32.
Allen-Baume V, Segui B, Cockcroft S. Current thoughts on the phosphatidylinositol transfer protein family. FEBS Lett 2002;531:74–80.
Yoder MD, Thomas LM, Tremblay JM, Oliver RL, Yarbrough LR, Helmkamp GM, Jr. Structure of a multifunctional protein. Mammalian phosphatidylinositol transfer protein complexed with phosphatidylcholine. J Biol Chem 2001;276:9246–52.
Tilley SJ, Skippen A, Murray-Rust J, Swigart P, Stewart A, Morgan CP, Cockcroft S, McDonald NQ. Structure-function analysis of human phosphatidylinositol transfer protein alpha bound to phosphatidylinositol. Structure 2004;12:317–26.
Schouten A, Agianian B, Westerman J, Kroon J, Wirtz KW, Gros P. Structure of apo-phosphatidylinositol transfer protein alpha provides insight into membrane association. EMBO J 2002;21:2117–21.
Segui B, Allen-Baume V, Cockcroft S. Phosphatidylinositol transfer protein-beta displays minimal sphingomyelin transfer activity and is not required for biosynthesis and trafficking of sphingomyelin. Biochem J 2002;366:23–34.
Morgan CP, Allen-Baume V, Radulovic M, Li M, Skippen AJ, Cockcroft S. Differential expression of a C-terminal splice variant of PITPβ lacking the constitutive-phosphorylated Ser262 that localises to the Golgi compartment. Biochem J 2006;398:411–21.
Li H, Tremblay JM, Yarbrough LR, Helmkamp GM, Jr. Both isoforms of mammalian phosphatidylinositol transfer protein are capable of binding and transporting sphingomyelin. Biochim Biophys Acta 2002;1580:67–76.
Holub BJ. The Mn2+-activated incorporation of inositol into molecular species of phosphatidylinositol in rat liver microsomes. Biochim Biophys Acta 1974;369:111–22.
Hunt AN, Skippen A, Koster G, Postle AD, Cockcroft S. Acyl chain-based molecular selectivity for HL60 cellular phosphatidylinositol and of phosphatidylcholine by phosphatidylinositol transfer protein α. Biochim Biophys Acta 2004;1686:50–60.
Brophy PJ, Burbach P, Nelemans SA, Westerman J, Wirtz KW, van Deenen LL. Evidence for the existence of different pools of microsomal phosphatidylinositol by the use of phosphatidylinositol-exchange protein. Biochem J 1978;174:413–20.
Thomas GMH, Cunningham E, Fensome A, Ball A, Totty NF, Troung O, Hsuan JJ, Cockcroft S. An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signalling. Cell 1993;74:919–28.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Cockcroft, S. (2009). Measurement of Phosphatidylinositol and Phosphatidylcholine Binding and Transfer Activity of the Lipid Transport Protein PITP. In: Larijani, B., Woscholski, R., Rosser, C. (eds) Lipid Signaling Protocols. Methods in Molecular Biology, vol 462. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-115-8_23
Download citation
DOI: https://doi.org/10.1007/978-1-60327-115-8_23
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-727-3
Online ISBN: 978-1-60327-115-8
eBook Packages: Springer Protocols