猪Ia相关恒定链定位基元突变对其定位功能的影响

Effect of the targeted mutation of sorting motifs on the localization of porcine Ia-associated invariant chain

目的研究猪Ia相关恒定链(Ii)胞质区2个亮氨酸基元(Leu7/Ile8和Met16/Leu17)周围氨基酸对基元内膜系统定位功能的影响。方法通过大引物PCR定点突变法,将2个亮氨酸基元及其周围氨基酸分别突变并导入pEGFP-C1真核表达质粒,获得21个突变Ii重组质粒。经LipofectamineTM2000将突变Ii重组质粒分别转染COS-7细胞,荧光显微镜检测突变Ii在细胞内的定位。结果 Leu 7/Ile 8和Met 16/Leu 17独立调控Ii分子的细胞内化。当保留其中一个亮氨酸基元,突变另一个亮氨酸基元周围氨基酸时,GFP-Ii融合蛋白或定位于内膜系统或分布于整个细胞。结论猪Ii链含有2个亮氨酸定位基元,亮氨酸基元的定位功能需要周围特定位置的氨基酸的支持。

Objective To investigate the effect of amino acids around two Leu-based motifs（Leu7/Ile8 and Met16/Leu17）in the cytosolic tail of porcine Ia-associated invariant chain（ Ii） on endomembrane system positioning function. Methods At first,two Leu-based motifs and amino acids around motifs were mutated through site-directed mutagenesis of the megaprimer PCR method,and the mutant genes were ligated to the vector pEGFP-C1,respectively. Twenty-one mutations of Ii recombinant plasmid were obtained and transiently transfected into the COS-7 cells by LipofectamineTM2000. Fluorescence microscopy was used to detect the intracellular localization of mutation Ii. Results Leu 7 /Ile 8 and Met 16 /Leu 17 independently mediated the intracellular localization of mutation Ii. When one leucine motif was kept and the amino acid around the other leucine motif was mutated,fusion protein GFP-Ii was located in the endomembrane system or distributed in the whole cell. Conclusion pig Ii comprises two independent sorting signals,and the functional Leu-based sorting signal requires specific neighboring residues.