Transbilayer Peptide Sorting Between Raft and Non-Raft Bilayers: Comparison of Detergent Extraction and Confocal Microscopy

¹ Duke University Medical Center

^* To whom correspondence should be addressed. E-mail: t.mcintosh{at}cellbio.duke.edu.

Submitted on March 4, 2005
Revised on April 7, 2005
Accepted on 16 May 2005

	Abstract

Membrane microdomains ("rafts") that sequester specific proteins and lipids are often characterized by their resistance to detergent extraction. Because rafts are enriched in sphingomyelin and cholesterol, raft bilayers are thicker and have larger area compressibility moduli than non-raft bilayers. It has been postulated that rafts concentrate proteins with long transmembrane domains (TMDs) because of "hydrophobic matching" between the TMDs and the thick raft bilayers. However, previous detergent extraction experiments with bilayers containing raft and non-raft domains have shown that the peptides P-23 and P-29, designed to have single TMDs matching the hydrocarbon thicknesses of DSMs and DRMs, respectively, are both localized to DSMs. These results imply that both peptides are preferentially located in non-raft domains. However, because the detergent solubilizes part of the bilayer, it has been unclear whether or not detergent extraction experiments provide an accurate indication of the location of peptides in intact bilayers. Here we use confocal microscopy to examine the distribution of these same peptides in intact bilayers containing both raft and non-raft domains. At 20 °C and 37 °C, P-23 and P-29 were both primarily localized in fluorescently labeled non-raft domains. These confocal results validate the previous detergent extraction experiments and demonstrate the importance of bilayer coheisve properties, compared to hydrophobic mismatch, in the sorting of these peptides that contain a single TMD.

Key Words: Bilayer Materials Properties, Cholesterol, Confocal Microscopy, Peptide Distribution, Rafts

This article has been cited by other articles:

				W. Rawicz, B. A. Smith, T. J. McIntosh, S. A. Simon, and E. Evans Elasticity, Strength, and Water Permeability of Bilayers that Contain Raft Microdomain-Forming Lipids Biophys. J., June 15, 2008; 94(12): 4725 - 4736. [Abstract] [Full Text] [PDF]

				J. Tong, L. Nguyen, A. Vidal, S. A. Simon, J. H. P. Skene, and T. J. McIntosh Role of GAP-43 in Sequestering Phosphatidylinositol 4,5-Bisphosphate to Raft Bilayers Biophys. J., January 1, 2008; 94(1): 125 - 133. [Abstract] [Full Text] [PDF]

				A. Pokorny, L. E. Yandek, A. I. Elegbede, A. Hinderliter, and P. F. F. Almeida Temperature and Composition Dependence of the Interaction of {delta}-Lysin with Ternary Mixtures of Sphingomyelin/Cholesterol/POPC Biophys. J., September 15, 2006; 91(6): 2184 - 2197. [Abstract] [Full Text] [PDF]