Tong Jihong, Nguyen Lam, Vidal Adriana, Simon Sidney A, Skene J H Pate, McIntosh Thomas J
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA.
Biophys J. 2008 Jan 1;94(1):125-33. doi: 10.1529/biophysj.107.110536. Epub 2007 Sep 7.
The lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) is critical for a number of physiological functions, and its presence in membrane microdomains (rafts) appears to be important for several of these spatially localized events. However, lipids like PIP(2) that contain polyunsaturated hydrocarbon chains are usually excluded from rafts, which are enriched in phospholipids (such as sphingomyelin) containing saturated or monounsaturated chains. Here we tested a mechanism by which multivalent PIP(2) molecules could be transferred into rafts through electrostatic interactions with polybasic cytoplasmic proteins, such as GAP-43, which bind to rafts via their acylated N-termini. We analyzed the interactions between lipid membranes containing raft microdomains and a peptide (GAP-43P) containing the linked N-terminus and the basic effector domain of GAP-43. In the absence or presence of nonacylated GAP-43P, PIP(2) was found primarily in detergent-soluble membranes thought to correspond to nonraft microdomains. However, when GAP-43P was acylated by palmitoyl coenzyme A, both the peptide and PIP(2) were greatly enriched in detergent-resistant membranes that correspond to rafts; acylation of GAP-43P changed the free energy of transfer of PIP(2) from detergent-soluble membranes to detergent-resistant membranes by -1.3 kcal/mol. Confocal microscopy of intact giant unilamellar vesicles verified that in the absence of GAP-43P PIP(2) was in nonraft microdomains, whereas acylated GAP-43P laterally sequestered PIP(2) into rafts. These data indicate that sequestration of PIP(2) to raft microdomains could involve interactions with acylated basic proteins such as GAP-43.
脂质磷脂酰肌醇-4,5-二磷酸（PIP₂）对多种生理功能至关重要，其在膜微结构域（脂筏）中的存在对于其中一些空间定位事件似乎很重要。然而，像PIP₂这样含有多不饱和烃链的脂质通常被排除在脂筏之外，脂筏中富含含有饱和或单不饱和链的磷脂（如鞘磷脂）。在这里，我们测试了一种机制，通过该机制多价PIP₂分子可以通过与多碱性细胞质蛋白（如GAP-43）的静电相互作用转移到脂筏中，GAP-43通过其酰化的N端与脂筏结合。我们分析了含有脂筏微结构域的脂质膜与含有GAP-43的连接N端和碱性效应结构域的肽（GAP-43P）之间的相互作用。在不存在或存在非酰化GAP-43P的情况下，PIP₂主要存在于被认为对应于非脂筏微结构域的去污剂可溶膜中。然而，当GAP-43P被棕榈酰辅酶A酰化时，肽和PIP₂都在对应于脂筏的去污剂抗性膜中大量富集；GAP-43P的酰化使PIP₂从去污剂可溶膜转移到去污剂抗性膜的自由能改变了-1.3千卡/摩尔。完整的巨型单层囊泡的共聚焦显微镜检查证实，在不存在GAP-43P的情况下，PIP₂位于非脂筏微结构域中，而酰化的GAP-43P将PIP₂横向隔离到脂筏中。这些数据表明，PIP₂隔离到脂筏微结构域可能涉及与酰化碱性蛋白（如GAP-43）的相互作用。
