Vergères G, Manenti S, Weber T, Stürzinger C
Department of Biophysical Chemistry, University of Basel, Switzerland.
J Biol Chem. 1995 Aug 25;270(34):19879-87. doi: 10.1074/jbc.270.34.19879.
Members of the myristoylated alanine-rich protein kinase C substrate (MARCKS) family are involved in several cellular processes such as secretion, motility, mitosis, and transformation. In addition to their ability to bind calmodulin and to cross-link actin filaments, reversible binding to the plasma membrane is most certainly an important component of the so far unknown functions of these proteins. We have therefore investigated the binding of murine MARCKS-related protein (MRP) to lipid vesicles. The partition coefficient, Kp, describing the affinity of myristoylated MRP for acidic lipid vesicles (20% phosphatidylserine, 80% phosphatidylcholine) is 5-8 x 10(3) M-1, which is only 2-4 times larger than the partition coefficient for the unmyristoylated protein. Interestingly, the affinity of MRP for acidic lipid membranes is 20-30-fold smaller than reported for murine MARCKS (Kim, J., Shishido, T., Jiang, X., Aderem, A. A., and McLaughlin, S. (1994) J. Biol. Chem. 269, 28214-28219). Since only a marginal binding could be observed with neutral phosphatidylcholine vesicles, we propose that electrostatic interactions are the major determinant of the binding of MRP to pure lipid membranes. Although the myristoyl moiety does not contribute drastically to the binding of MRP to vesicles, photolabeling experiments with a photoreactive phospholipid probe show that the fatty acid is embedded in the bilayer. The same membrane topology was found for bovine brain MARCKS. Since the relatively low affinity of MRP for vesicles is insufficient to account for a stable anchoring of the protein to cellular membranes, insertion of the myristoyl moiety into the bilayer might favor the interaction of MRP with additional factors required for the binding of the protein to intracellular membranes.
富含豆蔻酰化丙氨酸的蛋白激酶C底物（MARCKS）家族成员参与多种细胞过程，如分泌、运动、有丝分裂和转化。除了能够结合钙调蛋白和交联肌动蛋白丝外，与质膜的可逆结合无疑是这些蛋白质目前未知功能的重要组成部分。因此，我们研究了小鼠MARCKS相关蛋白（MRP）与脂质囊泡的结合。描述豆蔻酰化MRP对酸性脂质囊泡（20%磷脂酰丝氨酸，80%磷脂酰胆碱）亲和力的分配系数Kp为5 - 8×10³ M⁻¹，仅比未豆蔻酰化蛋白的分配系数大2 - 4倍。有趣的是，MRP对酸性脂质膜的亲和力比报道的小鼠MARCKS的亲和力小20 - 30倍（Kim, J., Shishido, T., Jiang, X., Aderem, A. A., and McLaughlin, S. (1994) J. Biol. Chem. 269, 28214 - 28219）。由于在中性磷脂酰胆碱囊泡中仅观察到微弱的结合，我们认为静电相互作用是MRP与纯脂质膜结合的主要决定因素。尽管豆蔻酰部分对MRP与囊泡的结合贡献不大，但用光反应性磷脂探针进行的光标记实验表明脂肪酸嵌入了双层膜中。牛脑MARCKS也发现了相同的膜拓扑结构。由于MRP对囊泡的相对低亲和力不足以解释该蛋白在细胞膜上的稳定锚定，豆蔻酰部分插入双层膜可能有利于MRP与该蛋白结合到细胞内膜所需的其他因子的相互作用。
