McIntosh Thomas J, Vidal Adriana, Simon Sidney A
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biophys J. 2003 Sep;85(3):1656-66. doi: 10.1016/S0006-3495(03)74595-0.
Specific proteins and lipids sequester to regions of cell membranes called rafts. Due to their high content of sphingomyelin (SM) and cholesterol, raft bilayers are thicker than nonraft bilayers and, at least at 4 degrees C, are resistant to Triton X-100 extraction. It has been postulated that rafts concentrate proteins with long transbilayer domains because of "hydrophobic matching" between the transbilayer domain and the thick bilayer hydrocarbon region. However, because the area compressibility and bending moduli of SM:cholesterol bilayers are larger than that of nonraft bilayers, there should be an energy cost to partition proteins or peptides into rafts. To determine the effects on peptide sorting of raft thickness and mechanical properties, we incorporated two transbilayer peptides (P-23, P-29) into bilayers composed of SM, dioleoylphosphatidylcholine, and cholesterol, separated detergent-soluble membranes (DSMs) from detergent-resistant membranes (DRMs), and measured their peptide and lipid compositions. P-23 and P-29 were designed to have transbilayer domains that matched the hydrocarbon thicknesses of DSMs and DRMs, respectively. At both 4 degrees C and 37 degrees C DSMs were enriched in dioleoylphosphatidylcholine and DRMs were enriched in SM and cholesterol. At both temperatures both P-23 and P-29 preferentially localized to DSMs, demonstrating the importance of bilayer mechanical properties relative to hydrophobic mismatch. However, at 37 degrees C significantly more P-29 than P-23 was located in DRMs, implying that hydrophobic matching played a role in peptide sorting at physiological temperature. These experiments demonstrate that the sorting of peptides as measured by detergent extraction is temperature-dependent and both bilayer mechanical properties and hydrophobic matching impact peptide distribution between DSMs and DRMs.
特定的蛋白质和脂质会聚集到细胞膜上称为脂筏的区域。由于其鞘磷脂（SM）和胆固醇含量高，脂筏双层比非脂筏双层更厚，并且至少在4℃时，对Triton X-100提取具有抗性。据推测，由于跨膜结构域与厚双层烃区域之间的“疏水匹配”，脂筏会聚集具有长跨膜结构域的蛋白质。然而，由于SM：胆固醇双层的面积压缩性和弯曲模量大于非脂筏双层，将蛋白质或肽分配到脂筏中应该存在能量成本。为了确定脂筏厚度和机械性能对肽分选的影响，我们将两种跨膜肽（P-23、P-29）掺入由SM、二油酰磷脂酰胆碱和胆固醇组成的双层中，将去污剂可溶膜（DSM）与去污剂抗性膜（DRM）分离，并测量它们的肽和脂质组成。P-23和P-29的设计使其跨膜结构域分别与DSM和DRM的烃厚度相匹配。在4℃和37℃时，DSM都富含二油酰磷脂酰胆碱，而DRM则富含SM和胆固醇。在这两个温度下，P-23和P-29都优先定位于DSM，这表明双层机械性能相对于疏水不匹配的重要性。然而，在37℃时，位于DRM中的P-29比P-23明显更多，这意味着疏水匹配在生理温度下的肽分选中起作用。这些实验表明，通过去污剂提取测量的肽分选是温度依赖性的，并且双层机械性能和疏水匹配都会影响DSM和DRM之间的肽分布。
