Cytosolic Localization of Mouse Peroxisomal Protein/ΔSKI Fused with Enhanced Green Fluorescent Protein into Chinese Hamster Ovary-K1 and P19 Cells

Abstract

Objective: Amino acid alignment analysis of deduced amino acid residues revealed a tripeptide(SKI) at the carboxy terminus of peroxisomal protein cDNA. In order to see the importanceof the above sorting signal, we have performed a site-directed mutagenesis to deleteSKI tripeptide and its transfection into CHO-K1 and P19 cells.Materials and Methods: In order to create the appropriate site-directed mutant, PCR wasdone with specific primers. Amplified PeP cDNAs either containing SKI or deleted ones wereconstructed downstream of EGFP cDNA under regulation of the cytomegalovirus (CMV)promoter in a pEGFP-C1 vector. Transfection of P19 and CHO cells was done with lipofectamine2000.Results: Gradient PCR showed that the best annealing temperature was 71.6 ºC. Transfectionof plasmids containing chimera of EGFP-PEP cDNAs into the CHO-K1 and P19 cellsshowed several punctuate structures, presumably peroxisomes, while SKI deletion showeda cytosolic mislocalization of the EGFP pattern.Conclusion: Taken together, these data strongly suggest that SKI, which is located at theC- terminus of the protein, is required for sorting this protein.

Keywords

• Peroxisomal Protein
• Peroxisome Targeting
• Signal Peptide