Identification and characterization of the human peroxin PEX3

The biogenesis of peroxisomes requires the interaction of several peroxins, encoded by PEX genes and is well conserved between yeast and humans, We ha ve cloned the human cDNA of PEX3 based on its homology to different yeast P EX3 genes, The deduced peroxin H sPEX3 is a peroxisomal membrane protein,vi th a calculated molecular mass of 42.1 kDa. We created N- and C-terminal ta gged PEX3 to assay its topology at the peroxisomal membrane by immunofluore scence microscopy. Our results and the one predicted transmembrane spanning region are in line with the assumption that H sPEX3 is an integral peroxis omal membrane protein with the N-terminus inside the peroxisome and the C-t erminus facing the cytoplasm. The farnesylated peroxisomal membrane protein PEX19 interacts with H sPEX3 in a mammalian two-hybrid assay in human fibr oblasts, The physical interaction could be confirmed by coimmunoprecipitati on of the two in vitro transcribed and translated proteins. To address the targeting of PEX3 to the peroxisomal membrane, the expression of different N- and C-terminal PEX3 truncations fused to green fluorescent protein (GFP) was investigated in human fibroblasts, The N-terminal 33 amino acids of PE X3 were necessary and sufficient to direct the reporter protein GFP to pero xisomes and seemed to be integrated into the peroxisomal membrane. The expr ession of a 1-16 PEX3-GFP fusion protein did not result in a peroxisomal lo calization, but interestingly, this and several other truncated PEX3 fusion proteins were also localized to tubular and/or vesicular structures repres enting mitochondria.