Transbilayer peptide sorting between raft and nonraft bilayers: comparisons of detergent extraction and confocal microscopy.
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 nonraft 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 nonraft domains have shown that the peptides P-23 and P-29, designed to have single TMDs matching the hydrocarbon thicknesses of detergent soluble membranes and detergent resistant membranes, respectively, are both localized to detergent soluble membranes. Those results imply that both peptides are preferentially located in nonraft 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 nonraft domains. At 20 degrees C and 37 degrees C, P-23 and P-29 were both primarily localized in fluorescently labeled nonraft domains. These confocal results validate the previous detergent extraction experiments and demonstrate the importance of bilayer cohesive properties, compared to hydrophobic mismatch, in the sorting of these peptides that contain a single TMD.
Vidal
Adriana
A
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
McIntosh
Thomas J
TJ
D003902:Detergents / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008560:Membrane Fluidity; D021962:Membrane Microdomains / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009038:Motion; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D044367:Phase Transition; D021381:Protein Transport
D003902:Detergents; D008051:Lipid Bilayers; D010455:Peptides