Blagoveshchenskaya A D, Hewitt E W, Cutler D F
MRC Laboratory for Molecular Cell Biology and Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, United Kingdom.
J Biol Chem. 1998 Oct 23;273(43):27896-903. doi: 10.1074/jbc.273.43.27896.
The 35-amino acid cytoplasmic tail of the adhesion receptor P-selectin is subdivided into stop transfer, C1 and C2 domains. It contains structural signals needed for targeting this protein to specialized secretory organelles and to lysosomes. Recently, using site-directed mutagenesis of horseradish peroxidase-P-selectin chimeras, we have uncovered a novel sequence within the C1 domain, KCPL, that mediates sorting from early, transferrin-positive endosomes to lysosomes and therefore operates as a positive lysosomal targeting signal (Blagoveshchenskaya, A. D., Norcott, J. P. , and Cutler, D. F. (1998) J. Biol. Chem. 273, 2729-2737). In the current study, we examined lysosomal targeting by both subcellular fractionation and an intracellular proteolysis assay and found that a balance of positive and negative signals is required for proper lysosomal sorting of P-selectin. First, we have found that within the sequence KCPL, Cys-766 plays a major role along with Pro-767, whereas Lys-765 and Leu-768 make no contribution to promoting lysosomal targeting. In addition, horseradish peroxidase-P-selectin chimeras were capable of acylation in vivo with [3H]palmitic acid at Cys-766, since no labeling of a chimera in which Cys-766 was replaced with Ala was detected. Second, analysis of mutations within the C2 domain revealed that substitution of two sequences, YGVF and DPSP, causes an increase in both lysosomal targeting and intracellular proteolysis suggesting the presence of lysosomal avoidance signals. The inhibition or promotion of lysosomal targeting resulted from alterations in endosomal sorting since internalization was not changed in parallel with lysosomal delivery. Analysis of the double mutants KCPL/YGVF or KCPL/DPSP revealed that although the positive lysosomal targeting signal operates in the early/sorting transferrin-positive endosomes, the negative lysosomal targeting (lysosomal avoidance) signals act at later stages of the endocytic pathway, most likely in late endosomal compartments.
黏附受体P-选择素的35个氨基酸的细胞质尾巴可细分为停止转移、C1和C2结构域。它包含将该蛋白靶向特定分泌细胞器和溶酶体所需的结构信号。最近，通过对辣根过氧化物酶-P-选择素嵌合体进行定点诱变，我们在C1结构域中发现了一个新序列KCPL，它介导从早期、转铁蛋白阳性的内体到溶酶体的分选，因此作为一个正向溶酶体靶向信号发挥作用（布拉戈韦申斯卡娅，A.D.，诺科特，J.P.，和卡特勒，D.F.（1998年）《生物化学杂志》273，2729 - 2737）。在当前的研究中，我们通过亚细胞分级分离和细胞内蛋白水解测定法研究了溶酶体靶向，发现P-选择素正确的溶酶体分选需要正负信号的平衡。首先，我们发现在序列KCPL中，Cys-766与Pro-767一起起主要作用，而Lys-765和Leu-768对促进溶酶体靶向没有贡献。此外，辣根过氧化物酶-P-选择素嵌合体在体内能够在Cys-766处被[3H]棕榈酸酰化，因为未检测到Cys-766被丙氨酸取代的嵌合体有标记。其次，对C2结构域内突变的分析表明，两个序列YGVF和DPSP的取代导致溶酶体靶向和细胞内蛋白水解都增加，这表明存在溶酶体回避信号。溶酶体靶向的抑制或促进是由于内体分选的改变，因为内化并没有与溶酶体递送同时发生变化。对双突变体KCPL/YGVF或KCPL/DPSP的分析表明，虽然正向溶酶体靶向信号在早期/分选转铁蛋白阳性内体中起作用，但负向溶酶体靶向（溶酶体回避）信号在胞吞途径的后期起作用，最有可能在晚期内体区室中起作用。
