The effect of site-directed mutagenesis of the ambient amino acids of leucine-based sorting motifs on the localization of chicken invariant chain

Two Leu residues and their ambient amino acid residues are known to exist in the cytosolic tail of chicken invariant chain (Ii), and these play an important role as motifs in mediating the sorting endocytic pathway. We performed 20 mutations via site-directed mutagenesis by the PCR megaprimer method to study the effect of some ambient amino residues of both Leu on the localization of chicken Ii. These mutated fragments were ligated to the vector pEGFP-C1. The recombinant plasmids were transiently transfected into COS-7 cells with Lipofectamine 2000. Furthermore, the fluorescence of located fusion proteins (green fluorescent protein-Ii) was observed with a fluorescence microscope. Our results indicated that 2 Leu-based motifs are required for chicken Ii intracellular localization, and both motifs independently mediate this function of the Ii. The other amino acid residues surrounding both Leu also influence Ii-induced endosomal vacuolation. In addition, we found that Pro(19), which is near the Val(17)-Leu(18) motif, was a key residue for chicken Ii intracellular localization. Not only is it critical for endocytic targeting to each Leu, but its unique mutation can also result in altering the function of chicken Ii.