INSULIN-DEGRADING ENZYME DOES NOT REQUIRE PEROXISOMAL LOCALIZATION FOR INSULIN DEGRADATION

Although considerable evidence implicates insulin-degrading enzyme (ID E) in the cellular metabolism of insulin in many cell types, its mecha nism and site of action are not clear. In this study, we have examined the relationship between insulin-degrading enzyme's peroxisomal locat ion and its ability to degrade insulin by mutation of its peroxisomal targeting signal (PTS), the carboxy terminal A/S-K-L tripeptide. Site- directed mutagenesis was used to destroy the peroxisomal targeting sig nal of human insulin-degrading enzyme by changing alanine to leucine ( AL.pts), leucine to valine (LV.pts), or by deleting the entire tripept ide (DEL.pts). The alanine or leucine mutants, when expressed in COS c ells, were indistinguishable from wild-type insulin-degrading enzyme w ith respect to size (110 kDa), amount of immunoreactive material, abil ity to bind insulin, in vitro activity, and cellular degradation of in sulin. In contrast, the deletion mutant was shorter in size (similar t o 0 kDa) and unable to bind the hormone. Thus, although the tripeptide at insulin-degrading enzyme's carboxy terminus appeared to confer enz yme stability, the conserved sequence was not required for insulin deg radation: Finally, an immunocytofluorescence study showed that, wherea s a significant amount of the wild-type protein was localized in perox isomes, none of the peroxisomal targeting mutants could be detected in these organelles. These findings indicate that insulin-degrading enzy me does not require peroxisomal localization for insulin degradation a nd suggest that this enzyme has multiple cellular functions.