Blagoveshchenskaya A D, Norcott J P, Cutler D F
Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, United Kingdom.
J Biol Chem. 1998 Jan 30;273(5):2729-37. doi: 10.1074/jbc.273.5.2729.
Signals controlling the intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. P-selectin is a type I membrane protein receptor for leukocytes, acting during the inflammation response. Heterologous expression experiments have demonstrated that its 35-residue cytoplasmic tail contains signals for targeting to synaptic-like microvesicles, dense-cored granules, and lysosomes. We have examined the lysosomal targeting information present within the cytoplasmic tail by site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras followed by transient transfection in H.Ep.2 cells. Assaying lysosomal targeting by subcellular fractionation as well as intracellular proteolysis, we have discovered a novel lysosomal targeting signal, KCPL, located within the C1 domain of the cytoplasmic tail. Alanine substitution of this tetrapeptide reduced lysosomal targeting to the level of a tailless horseradish peroxidase-P-selectin chimera, which was previously found to be deficient in both internalization and delivery to lysosomes. A proline residue within this lysosomal targeting signal makes a major contribution to the efficiency of lysosomal targeting. A diaminobenzidine density shift procedure established that chimeras with an inactivated KCPL sequence are present within transferrin-positive compartments. Such a mutant also displays an increased level of expression at the plasma membrane. Our results indicate that the sequence KCPL within the cytoplasmic tail of P-selectin is a structural element that mediates sorting from endosomes to lysosomes.
控制许多膜蛋白细胞内靶向定位的信号，以短序列形式存在于它们的胞质结构域中。P-选择素是一种白细胞的I型膜蛋白受体，在炎症反应中发挥作用。异源表达实验表明，其35个氨基酸残基的胞质尾巴含有靶向突触样微囊泡、致密核心颗粒和溶酶体的信号。我们通过对辣根过氧化物酶-P-选择素嵌合体进行定点诱变，然后在H.Ep.2细胞中瞬时转染，研究了胞质尾巴中存在的溶酶体靶向信息。通过亚细胞分级分离以及细胞内蛋白水解来检测溶酶体靶向定位，我们发现了一个新的溶酶体靶向信号KCPL，它位于胞质尾巴的C1结构域内。将这个四肽中的丙氨酸进行替换，可将溶酶体靶向定位降低到无尾辣根过氧化物酶-P-选择素嵌合体的水平，而之前发现该嵌合体在内化和向溶酶体的递送方面均存在缺陷。这个溶酶体靶向信号中的一个脯氨酸残基对溶酶体靶向定位的效率起主要作用。二氨基联苯胺密度转移程序确定，具有失活KCPL序列的嵌合体存在于转铁蛋白阳性区室中。这样的突变体在质膜上也表现出表达水平的增加。我们的结果表明，P-选择素胞质尾巴中的KCPL序列是一个介导从内体到溶酶体分选的结构元件。
