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

OBJECTIVE:To investigate the effect of amino acids around two Leu-based motifs (Leu 7/Ile 8 and Met 16/Leu 17) 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 Lipofectamine(TM) 2000. 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.

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