Peroxisome targeting of porcine 17 beta-hydroxysteroid dehydrogenase type IV/D-specific multifunctional protein 2 is mediated by its C-terminal tripeptide AKI

The product of the porcine HSD17B4 gene is a peroxisomal 80 kDa polypeptide containing three functionally distinct domains. The N-terminal part reveal s activities of 17 beta-estradiol dehydrogenase type IV and D-specific 3-hy droxyacyl CoA dehydrogenase, the central part shows D-specific hydratase ac tivity with straight and 2-methyl-branched 2-enoyl-CoAs. The C-terminal par t is similar to sterol carrier protein 2. The 80 kDa polypeptide chain ends with the tripeptide AKI, which resembles the motif SKL, the first identifi ed peroxisome targeting signal PTS1. So far AKI, although being similar to the consensus sequence PTS1, has neither been reported to be present in mam malian peroxisomal proteins, nor has it been shown to be functional. We inv estigated whether the HSD17B4 gene product is targeted to peroxisomes by th is C-terminal motif. Recombinant human PTS1 binding protein Pex5p interacte d with the bacterially expressed C-terminal domain of the HSD17B4 gene prod uct. Binding was competitively blocked by a SKL-containing peptide. Recombi nant deletion mutants of the C-terminal domain lacking 3, 6, and 14 amino a cids and presenting KDY, MIL, and IML, respectively, at their C-termini did not interact with Pex5p. The wild-type protein and mutants were also trans iently expressed in the HEK 293 cells. Immunofluorescence analysis with pol yclonal antibodies against the C-terminal domain showed a typical punctate peroxisomal staining pattern upon wild-type transfection, whereas all mutan t proteins localized in the cytoplasm. Therefore, AKI is a functional PTS1 signal in mammals and the peroxisome targeting of the HSD17B4 gene product is mediated by Pex5p. J. Cell. Biochem. 73:70-78, 1999. (C) 1999 Wiley-Liss , Inc.