Sorting analysis of mouse peroxisomal protein by in vitro studies

The recently cloned peroxisomal matrix protein (PeP), contains two hydrophobic domains at 12-31 and 152-169 amino acid residues and a fibronectin type III (FnIII) domain between residues 31-114. To understand the importance of the above mentioned domains in peroxisome sorting of PeP, site-directed mutagenesis was performed to delete these domains separately. Amplified mutants of PEP cDNA were constructed downstream of enhanced green fluorescent protein (EGFP) cDNA for transfection into the chinese hamster ovary (CHO) K1 and P19 cell lines. Upon their transfections, numerous green fluorescent punctuate structures, superimposable on those punctuates stained with the anti-catalase antibody, appeared in the cells. Thus, the aforementioned domains do not exert the targeting signal activity for the peroxisomal sorting of PeP.