Insulin-Degrading Enzyme Does Not Require ...

0013-7227/97/$03.00/0 Endocrinology Copyright © 1997 by The Endocrine Society

Vol. 138, No. 8 Printed in U.S.A.

Insulin-Degrading Enzyme Does Not Require Peroxisomal Localization for Insulin Degradation* ´ RIE CHESNEAU†‡, RACHEL K. PERLMAN†, WENLU LI, VALE ´ KELLER, AND MARSHA RICH ROSNER GILBERT-ANDRE Ben May Institute for Cancer Research (V.C., R.K.P., M.R.R.), The University of Chicago, Chicago, Illinois 60637; Department of Pharmacology (W.L., G.-A.K.), Genetech, Inc., San Francisco, California 94080; Columbia University College of Physicians and Surgeons (R.K.P.), New York, New York 10032 ABSTRACT Although considerable evidence implicates insulin-degrading enzyme (IDE) in the cellular metabolism of insulin in many cell types, its mechanism and site of action are not clear. In this study, we have examined the relationship between insulin-degrading enzyme’s peroxisomal location 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 signal of human insulin-degrading enzyme by changing alanine to leucine (AL.pts), leucine to valine (LV.pts), or by deleting the entire tripeptide (DEL.pts). The alanine or leucine mutants, when expressed in COS cells, were indistinguishable from wild-type insulin-degrading enzyme with respect to size (110 kDa), amount of

immunoreactive material, ability to bind insulin, in vitro activity, and cellular degradation of insulin. In contrast, the deletion mutant was shorter in size (;0 kDa) and unable to bind the hormone. Thus, although the tripeptide at insulin-degrading enzyme’s carboxy terminus appeared to confer enzyme stability, the conserved sequence was not required for insulin degradation. Finally, an immunocytofluorescence study showed that, whereas a significant amount of the wild-type protein was localized in peroxisomes, none of the peroxisomal targeting mutants could be detected in these organelles. These findings indicate that insulin-degrading enzyme does not require peroxisomal localization for insulin degradation and suggest that this enzyme has multiple cellular functions. (Endocrinology 138: 3444 – 3451, 1997)

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(8 –10). Monoclonal antibodies to IDE can specifically inhibit insulin degradation in HepG2 cells (11), and the protease can be cross-linked to insulin in intact cells (12). Moreover, its overexpression in COS cells increases the rate of intracellular insulin degradation severalfold, indicating that IDE catalyzes a rate-determining step in insulin degradation (13). In antigen presenting cells, IDE has also been proposed to mediate the processing of insulin epitopes for helper T cells (14). Although the evidence for the role of IDE in degrading insulin is strong, there is increasing evidence that the metalloprotease may have other cellular substrates as well. Indeed, in vitro studies have shown that IDE can degrade the related growth factors insulin-like growth factor I and II (15), as well as transforming growth factor (16). Other in vitro substrates that have more recently been identified are atrial natriuretic peptide (17) and oxidatively damaged hemoglobin (18). Although the biological role of IDE remains a fundamental question, its high degree of evolutionary conservation further supports the idea that it must have important functions. Rat, Drosophila, and bacterial homologs have been cloned that have 95%, 47%, and 26% identity, respectively, with the complementary DNA (cDNA)-deduced amino acid sequence of the human enzyme (19 –22). More recently, two members of the IDE family have been identified in yeast Saccharomyces cerevisiae (23), and three in Caenorhabditis elegans. In addition to an overall homology, IDE contains several conserved functional motifs. We recently verified that the conserved HXXEH sequence in the human IDE is a zinc binding motif that serves as the core of IDE’s active site (5, 24). Comparison of the amino acid sequences of the human, rat and Drosophila IDEs has also revealed a conserved car-

NSULIN IS ONE of a few known factors required by virtually all cell types for optimal growth and proliferation (1), but many of its effects are tissue- or cell-type specific (2). Insulin signaling is initiated when the hormone binds to its receptor on the cell surface. After binding, the receptorinsulin complex is internalized via receptor-mediated endocytosis, and the internalized insulin is eventually either degraded or released intact (reviewed in Ref. 3). Higher concentrations of insulin are associated with higher percentages of internalized insulin being released intact (4). The degradation of insulin, therefore, may play a role in termination of the signal and/or clearance of the circulating hormone. The insulin-degrading enzyme (IDE; EC 3.4.22.11) is an evolutionarily conserved neutral thiol metalloprotease able to degrade insulin in vitro with high specificity and very low Km (5). Numerous experiments suggest that IDE is the principal enzyme controlling insulin degradation in many cell types. The sites at which the purified enzyme cleaves insulin in vitro are consistent with the insulin degradation products found in intact liver (6) and cultured cells (7). Inhibitors of IDE block insulin degradation in a number of cell types

Received February 27, 1997. Address all correspondence and requests for reprints to: Marsha Rich Rosner, Ben May Institute for Cancer Research, University of Chicago, 5841 South Maryland Avenue, MC 6027, Chicago, Illinois 60637. E-mail: [email protected]. * This work was supported by a gift from the Cornelius Crane Trust (to M.R.R.). † Equal author contribution. ‡ Recipient of fellowship from the Association pour la Recherche contre le Cancer.

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IDE DEGRADES INSULIN OUTSIDE OF PEROXISOMES

boxy-terminal peroxisomal targeting signal, A/S-K-L (25, 26). Peroxisomes are single-membrane-bound organelles involved in the generation and degradation of H2O2, in plasmalogen synthesis, cholesterol and bile acid synthesis, purine and amino acid catabolism, glyoxylate utilization, and prostaglandin metabolism (27, 28). Peroxisomal proteins are synthesized on free polysomes and are directed into the organelle posttranslationally by at least two pathways dependent on distinct peroxisomal targeting signals (PTS1 and PTS2). The PTS1 signal is a C-terminal tripeptide (SKL or a variant), whereas PTS2 is a NH2-terminal peptide (29). IDE from human, rat, and Drosophila contains a conserved PTS1 (A/S-K-L), but the homologous protease from Escherichia coli does not. A number of studies have now shown that IDE is indeed located in peroxisomes in mammalian cells. In subcellular fractionation studies using rat liver, IDE cosedimented with peroxisomal markers (30). Recently, we have established stable Ltk2 cell lines in which induction of IDE expression results in increased cellular insulin degradation (31). Immunofluorescence and immunocryoelectron microscopy revealed that IDE in these cells was localized primarily in peroxisomes, although a lesser amount was found in the cytosol. Similar observations have been recently made in transfected CHO cells (32). In the present study, we have made changes in the peroxisomal targeting signal of IDE that block IDE transport to peroxisomes. Analysis of these mutants showed that IDE does not require peroxisomal localization for insulin degradation. The enzyme may thus participate in several distinct physiological processes. Materials and Methods Materials Restriction enzymes were purchased from either Life Technologies, Inc. (Grand Island, NY) or New England Biolabs (Beverly, MA). Insulin and BSA were purchased from Sigma (St. Louis, MO). Disuccinimidyl suberate was from Pierce. Prestained standard SDS-PAGE molecular weight markers were purchased from Bio-Rad (Richmond, CA). Nytran membranes were purchased from Schleicher and Schuell (Keene, NH). 125 I-Insulin (A14 monoiodinated receptor grade, 2200 Ci/mmol) was purchased from DuPont-New England Nuclear (Boston, MA). 125I-Protein A was a gift from Geoffrey Green of the Ben May Institute or purchased from DuPont-NEN. Oligonucleotides were obtained from Operon (Alameda, CA) or from Paul Gardner of the Howard Hughes Institute of the University of Chicago. Polyclonal antibodies to IDE (antiserum 2BS) were produced by immunizing rabbits with trpE fusion proteins containing amino acids 118 to 508 of human IDE (33). Monoclonal antibodies to human IDE (antibody 31H7) were a gift from Richard Roth of Stanford University (11). Monoclonal antibodies to the hemagglutinin1 (HA) epitope were purchased from Berkely Antibody Company (Richmond, CA).

General techniques COS and Ltk2 cells were grown in DMEM supplemented with 10% FBS (Life Technologies, Inc.). Protein concentrations were estimated by a modified Bradford (34) assay (Bio-Rad). SDS-PAGE was done using the conditions of Laemmli (35).

Production of mutated plasmids pCMVhIDE was constructed by inserting the 3.4 kb cDNA for human insulin-degrading enzyme into pCMVo as previously described (13). The PTS mutants were created using the unique site elimination mutagenesis kit from Pharmacia as described (36). Briefly, the kit uses a two-primer

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system to generate site-specific mutations (37). The first primer carried the desired mutation, either 3106GC to CT for AL.pts, 3112C to G for LV.pts, and a deletion of nucleotides 3106 to 3114 for DEL.pts. (The nucleotide positions refer to the sequence published in Ref. 20).) The second primer carried a mutation in an XbaI site, which was a unique site in the pCMVo vector and was in a nonessential region of the vector. Both mutations were incorporated simultaneously by primer-directed DNA polymerization using pCMVhIDE as a template. After amplification in bacteria, plasmids were screened by digestion with XbaI. Clones that had lost this unique site were then sequenced to choose cDNAs with the appropriate mutations. The plasmids containing the hemagglutinin (HA) epitope-tagged wtIDE and AL.pts were constructed as previously described (36).

Transfection of DNA DNA for transfection was purified by CsCl gradient centrifugation followed by RNase digestion and centrifugation through 1 m NaCl to remove oligoribonucleotides (38). Mutant plasmids were transfected with pSV2CAT into COS cells by calcium phosphate precipitation as previously described (13). Cells were harvested or assayed 36 – 48 h after glycerol shock. For the immunocytofluorescence study, COS and Ltk2 cells were replated 6 h after glycerol shock and grown on glass coverslips for another 20 h.

Preparation of cell extracts and Western blot analysis Preparation of cell extracts and Western blotting were performed as previously described (24). For Western blot analysis, 50 mg of each cell extract were electrophoresed under reducing conditions, and proteins were blotted onto nitrocellulose with a HoefferSem-Phor transfer apparatus. Blots were probed with 1/200 dilutions of antiserum 2BS (33) or preimmune serum.

Insulin degradation assays Cellular degradation of insulin was assayed as previously described (24). Briefly, COS cells on 100-mm plates were washed once with isotonic PBS, preincubated 30 min in 4 ml binding buffer (1 mg/ml BSA in DMEM) and then switched to 4 ml of 100 pm 125I-insulin in binding buffer. Triplicate 150 ml aliquots were taken at each time points, and undegraded insulin was precipitated by addition of one volume of 30% trichloroacetic acid. Acid-soluble label at time zero was taken as a blank. To assay conditioned medium, the binding buffer was collected after 30 min (before addition of label) and was centrifuged to remove any detached cells. Then, 100 pm 125I-insulin was added to the conditioned medium and 150 ml aliquots were taken after the indicated time of incubation at 37 C. Undegraded insulin was precipitated by the addition of one volume of 30% trichloroacetic acid. In vitro assay of IDE activity was performed as previously described (13), using aliquots of cellular extract containing 1 mg protein.

Affinity labeling Insulin affinity labeling was performed as previously described (36). Briefly, aliquots of COS cell extracts containing equal amounts of total protein were added to 0.5 nm 125I-insulin, 50 mm HEPES (pH7.5), 50 mm NaCl, and 1 mg/ml BSA in a total volume of 100 ml. Nonspecific labeling was assessed by addition of 3.3 mm unlabeled insulin. 5 ml of 3 mg/ml disuccinimidyl suberate in dimethyl sulfoxide was added to each tube. After 60 min incubation on ice, the cross-linking reactions were terminated by addition of SDS-gel electrophoresis sample buffer. The samples were then heated and electrophoresed, and cross-linked proteins were visualized by autoradiography of the dried gel.

Immunocytofluorescence microscopy Fixation and immunofluorescence labeling of transfected COS and Ltk2 cells were performed by a modification of the method previously described (39). Briefly, cells plated on coverslips were fixed for 30 min in 10% buffered formalin, permeabilized for 5 min with 0.2% Triton in 3% buffered formalin, and blocked for 30 min with 5% dry milk in PBS pH 7.2. 31H7 antibody to IDE (11) was used at the 75 mg/ml final

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concentration. Mouse anti-HA antibodies were used at the 1/1000 final dilution. In double labeling experiments, a rabbit antibovine catalase antibody (1/200 dilution; Biodesign, Kennebunkport, ME) or a guinea pig anti-rat peroxisomal membrane protein (PMP70) antibody (1/200 dilution) were used simultaneously with the specific mouse antibody against human IDE, 31H7, or the mouse anti-HA antibody. The secondary antibodies were Cy3-conjugated donkey to rabbit or guinea pig IgG and fluorescein-conjugated donkey antimouse (Jackson Immunoresearch Laboratories, Inc., West Grove, PA). COS cells transfected with the pCMVo vector alone were used as a negative control. Cells were photographed on the same plane of focus with a Leitz (Wetzlar, Germany) Aristoplan microscope using appropriate filters for Cy3 and fluorescein.

Results Generation of peroxisomal targeting mutants

Three mutations of the A-K-L peroxisomal targeting signal of human IDE were generated by site-specific mutagenesis using primer-directed DNA polymerization on a plasmid template. A pCMVo expression vector containing the 3.4-kb cDNA of human IDE was used as the template. Specifically, one mutation was made that changed the alanine to leucine (AL.pts), one mutation was made that changed the leucine to valine (LV.pts), and one mutation was made that deleted the entire tripeptide (DEL.pts.) Previous studies have demonstrated that these mutations of the peroxisomal targeting signal abolish the import of peroxisomal proteins transiently expressed in CV-1 monkey kidney cells (25). Expression of wild-type IDE and peroxisomal targeting mutants in transfected COS cells

We have previously used this pCMVo vector, which has a cytomegalovirus promoter and the SV40 origin, to transiently express IDE in COS cells. Those experiments demonstrated that overexpression of IDE results in increased cellular degradation of insulin (13). In the present experiments, wild-type and mutant enzymes were transiently expressed in COS monkey kidney cells by transfection of pCMVo vectors containing the appropriate cDNA. The pCMVo vector alone was used as a negative control. Western blotting with anti-IDE antibody (2BS antibody) was used to confirm expression of the transfected genes. An immunoreactive band at the correct molecular weight for IDE (110,000) was increased severalfold over the pCMVo

FIG. 1. Production of immunoreactive protein in COS cells transfected with wtIDE and peroxisomal targeting mutants. Extracts from transfected cells were prepared, electrophoresed, and Western blotted with antiserum 2BS as described under Materials and Methods. Fifty micrograms were used for each extract. Bands were visualized by autoradiography of 125I-protein A. Arrows with numbers (in kDa) indicate the position of prestained molecular mass markers.

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control in extracts of cells transfected with wild-type IDE1 (wtIDE), AL.pts, or LV.pts (Fig. 1). Interestingly, the deletion mutant shows a lower molecular weight protein that is reduced in amount relative to the other overexpressed IDEs. This band is smaller than that which we occasionally see at 100,000, and which has been determined to represent a degradation product of IDE. The faint band at 110 kDa in the DEL.pts lane is endogenous IDE and is identical in size and amount to the band in the pCMVo lane. These results suggest that the AL.pts and LV.pts mutants are not altered in stability, but that the deletion mutation appears to affect the stability of the protein. A similar result was obtained with an independent clone expressing the deletion mutant, indicating that the result is not an artifact of clonal variation (data not shown.) Insulin degrading activity and binding ability of peroxisomal targeting mutants

We have previously shown that transient overexpression of wtIDE in COS cells results in increased degradation of insulin via an intracellular pathway (13). To determine whether the peroxisomal A-K-L targeting signal is required for IDE to degrade insulin, we measured the ability of mutant-transfected COS cells to hydrolyze cellular insulin relative to control cells. Cells were incubated in binding buffer for 30 min and then switched to fresh buffer containing 100 pm 125I-insulin. Degradation was assayed as described in Materials and Methods. We observed a 2- to 3-fold increase in the rate of cellular degradation of insulin in cells transfected with wtIDE, as compared with those transfected with vector alone (Fig. 2). While both AL.pts and LV.pts increased insulin degradation to the same extent as the wild-type enzyme, the

FIG. 2. Degradation of insulin by COS cells transfected with wtIDE or peroxisomal targeting mutants. COS cells were transfected with pCMVo, wtIDE, and the three mutants of the peroxisomal targeting signal. Forty hours after transfection, cells were preincubated for 30 min in binding buffer and then switched to fresh binding buffer containing 100 pM 125I-insulin. Cells were incubated for the indicated times and the extent of insulin degradation was quantitated by trichloroacetic acid-precipitation as described in Materials and Methods. The results are shown as means 6 SD of triplicate determinations. Where not shown, the error bars are smaller than the plot symbol.


deletion mutant did not increase the rate of insulin degradation above that seen with vector alone. Because the stress of transfection can result in some nonspecific leakage of proteins into the cell medium, we assayed conditioned medium to determine whether AL.pts and LV.pts are really degrading insulin intracellularly. We collected conditioned medium (binding buffer without labeled insulin) that had been incubated with the cells for 30 min and assayed it for insulin-degrading activity in parallel to the transfected cells (Fig. 3). Insulin degradation in the conditioned medium did not account for more than 1/5 of the total insulin degradation and was comparable with that observed in cells expressing wtIDE. These results indicate that two mutations, AL.pts and LV.pts, do not affect the ability of the enzyme to degrade insulin in cells. We also assayed the ability of all three mutants to catalyze the in vitro degradation of insulin. Insulin-degrading activity was increased 4- to 6-fold in extracts from cells transfected with wtIDE, AL.pts, or LV.pts (Fig. 4). In contrast, extracts from cells transfected with DEL.pts showed the same specific activity as those from cells transfected with pCMVo. These results demonstrate that deletion of the terminal tripeptide results in loss of enzyme activity, whereas altering one amino acid in the conserved peroxisomal targeting tripeptide had no effect on the catalytic activity of the mutant proteins. Affinity labeling with 125I-insulin was used to assess the ability of the mutants to bind this substrate. Cell extracts from each transfectant were incubated with 125I-insulin at 0 C and covalently cross-linked using disuccinimidyl suberate. As expected, extracts of cells transfected with wild-type IDE had increased labeling as compared with extracts from the pCMVo control (Fig. 5). Extracts from the AL.pts and LV.pts mutants also showed an increase in cross-linking to

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FIG. 4. Insulin-degrading activity in extracts from cells transfected with wtIDE or peroxisomal targeting mutants of IDE. Extracts from transfected cells were prepared and assayed for insulin-degrading activity as described under Materials and Methods using 1 mg of total cell extract per assay. Results are shown as means 6 SD of triplicate determinations.

FIG. 5. Cross-linking of 125I-insulin to peroxisomal targeting mutants. Extracts were prepared and affinity-labeled in the presence of 0.5 nM 125I-insulin with or without and excess of unlabeled insulin. Cross-linked products were separated by SDS-gel electrophoresis and visualized by autoradiography. The Mr of the observed bands was about 110,000 by comparison with prestained markers. 125

I-insulin, suggesting that the sequence A-K-L is not directly involved in binding of substrate. The DEL.pts mutant did not cross-link to insulin at all, indicating that the protein visualized by Western blotting (Fig. 1), although close in size to wild-type enzyme, is incapable of binding insulin. Because this protein appears to be greater than 90 kDa by comparison with prestained molecular weight markers, it seems likely that improper folding of the protein has resulted in loss of ability to recognize its substrate.

Immunolocalization of the overexpressed peroxisomal targeting mutant and wild-type IDEs

FIG. 3. Degradation of insulin in conditioned medium from COS cells transfected with wtIDE or peroxisomal targeting mutants. Cells were transfected with pCMVo, wtIDE, or with one of three peroxisomal targeting mutants of IDE. Before assaying cellular degradation, cells were preincubated with binding buffer for 30 min. This buffer was then transferred to a fresh plate and 100 pM 125I-insulin was added. Degradation was measured at the times indicated. Results are shown as means 6 SD of triplicate determinations. Where not shown, the error bars are smaller than the plot symbol.

To verify that the three mutations of IDE’s peroxisomal targeting signal effectively abolished the import of the protease into peroxisomes, the cellular localization of wild-type and mutant IDEs was determined by immunocytofluorescence and confocal microscopy analysis. Figure 6A, which shows immunofluorescent labeling of the LV.pts, illustrates one of the labeling patterns obtained for both single mutants, LV.pts and AL.pts. In contrast to the punctate labeling of catalase (Fig. 6B), the IDE labeling in

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FIG. 6. Micrographs of COS cells transfected with LV.pts, HA-tagged AL.pts, and wtIDE. COS cells transfected with either LV.pts (A to C), HA-tagged AL.pts (D to F) or wtIDE (G and H) were fixed and double labeled, with either 31H7 or anti-HA antibodies, and anticatalase or anti-PMP70 antibodies, as described in Materials and Methods. A, D, and G, Labeling of LV.pts with the anti-IDE antibody 31H7, HA-tagged AL.pts with the anti-HA antibody, and wtIDE with 31H7 respectively. B and E, Immunolabeling of catalase; H, labeling of the peroxisomal membrane protein PMP70. C and F, Nomarski views of the stained cells. The bars in C, F, and H correspond to 10 mm.


these cells was diffuse. Cytosolic staining was observed in all transfected cells. Additionally, nuclear staining could sometimes be observed, but its intensity seemed to vary with the relative level of protein overexpression. No colocalization was observed by confocal microscopy between catalase and the peroxisomal targeting mutants of IDE, indicating that IDE was not translocated to the peroxisome. Similar results were obtained when the peroxisomal membrane protein PMP70 (40) was used as a marker instead of catalase (data not shown). To rule out any immunolabeling artifacts, similar experiments were performed on COS cells transfected with an HA epitope-tagged AL.pts construct. The HA-tagged mutant of IDE was detected using an antibody specifically directed against the hemagglutinin epitope. We have previously shown that, when inserted behind the second ATG, the hemagglutinin sequence does not modify the enzyme’s ability to bind insulin or its degrading activity, suggesting that the tagged endopeptidase was functional (36). The HA-tagged AL.pts appeared to have the same subcellular localization as the corresponding untagged protein. Figure 6D, which shows immunofluorescent labeling of the HA-tagged AL.pts, illustrates the second labeling pattern obtained for AL.pts and LV.pts. In contrast to the punctate catalase staining (Fig. 6E), both cytoplasm and nucleus appeared strongly labeled in this group of cells. The absence of colocalization with catalase was verified by confocal microscopy analysis. The labeling of cells expressing DEL.pts was essentially nuclear (data not shown). In cells expressing moderate levels of wtIDE, the enzyme was localized mostly to the peroxisomes as shown by the double labeling of wtIDE and the peroxisomal marker PMP70 on the same cells (Figs. 6, G and H). The same results were obtained using an antibody to catalase (not shown). In addition, a faint immunofluorescence for IDE could also be seen in the cytoplasm (Fig. 6G). However, in cells overexpressing wtIDE, in addition to peroxisomes and the cytoplasm, the enzyme could be observed sometimes in the nucleus as noted above for the peroxisomal targeting mutants (data not shown). Taken together, these data show that both point mutations of the human IDE’s peroxisomal targeting signal, as well as its deletion, abolished the import of the protein into peroxisomes. Finally, in contrast to transiently transfected COS cells, only minimal nuclear staining was previously observed in Ltk2 cells stably expressing wtIDE under an inducible promoter (31). To check if the strong nuclear labeling sometimes obtained in COS cells was cell type specific, we also transiently expressed wtIDE, HA epitope-tagged wtIDE, and AL.pts into Ltk2 cells. The protein was detected using the 31H7 anti-IDE monoclonal antibody or the anti-HA antibody, and the same labeling pattern as in COS cells was obtained with all three constructs (not shown). Although a physiological role for the nuclear IDE cannot be excluded at this point, these data suggest that overexpression of IDE in transiently transfected cells may also lead to its nuclear accumulation possibly by nonspecific translocation through the nuclear pores. This observation has been made previously in mammalian cells overexpressing peroxisomal proteins, including firefly luciferase (41), and in yeast (42, 43).

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Discussion

IDE was recently localized primarily to peroxisomes by immunofluorescence and immunoelectron microscopy using a cell line stably expressing IDE on an inducible vector (31). Induction of IDE expression increased insulin degradation by these cells. In the present study, two mutations of human IDE’s peroxisomal targeting signal abolished its import into peroxisomes but did not affect the ability of IDE to degrade insulin, either in vitro or in intact cells. This finding shows that IDE can degrade insulin without being localized to peroxisomes. The absence of a full-sized IDE and the slight presence of a lower molecular weight protein in extracts from cells transfected with the deletion mutant (DEL.pts) suggest that this mutant is rapidly degraded in the cytosol. It is surprising that the deletion of just three amino acids causes such a dramatic change in protein stability, whereas the two singly mutated IDEs (AL.pts and LV.pts) are stable. We cannot distinguish at this point whether this effect is cell-type specific, or more general. However, one possible explanation is that alanine or leucine mutants maintain association with molecules that protect them from degradation, although they are still incapable of peroxisomal translocation. There is evidence that cytosolic SKL binding factors such as the 70-kDa heat shock proteins are involved in peroxisomal import (27, 44). Recently, a cytosolic 70-kDa protein has been found to be associated with the cytosolic pool of IDE in hepatoma HepG2 cells, and has been proposed to maintain the dual cytosolic and peroxisomal pools of IDE in a stable equilibrium (45). Yet another possibility is that deletion of the COOH-terminal tripeptide of IDE somehow alters protein folding or conformation, resulting in reduced stability. Interestingly, whereas the singly mutated proteins were found in both the cytoplasm and nucleus, the deletion mutant was almost exclusively nuclear (data not shown). This observation reinforces the idea that the deletion mutant was not stable in the cytosol, and was rapidly degraded in this cell compartment. Although wild-type IDE is primarily detected in peroxisomes, the protease is also present and may function in the cytosol. Significant amounts were detected in the cytosol of Ltk2 cells stably expressing inducible IDE (31), in CHO cells overexpressing IDE (32), and both in transiently transfected COS and Ltk2 cells in the present study. Other peroxisomal proteins, including catalase, have been reported to be present also in the cytosol. Recent evidence that guinea pig liver peroxisomal and cytoplasmic catalases are two related but distinct proteins that differ in size and amino acid composition (46) suggests that catalase may function in both subcellular compartments. Several arguments support the hypothesis that IDE also functions in the cytosol. First, the relative proportion of the protein in peroxisomes vs. the cytosol seems to be cell-type specific. In HepG2 cells, IDE was found to be mainly cytosolic, and the cytoplasmic pool appeared unchanged in cells undergoing peroxisomal proliferation (45). Secondly, IDE is fairly abundant in red blood cells (20) that lack peroxisomes. In the present study, the observation that the extent of insulin degradation appears to be unaffected by IDE peroxisomal localization is consistent with IDE degrading the hor-

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mone in the cytoplasm. Indeed, the alanine and leucine mutants were able to degrade cellular insulin as well as the wild-type endopeptidase, which we have previously shown to act intracellularly (13). Because insulin degradation does not require IDE to be in peroxisomes, it is likely that IDEmediated insulin degradation does not occur in these organelles. If this is the case, then one might expect the level of insulin degradation to be higher in cells expressing the singly mutated IDEs than in those expressing the wild-type enzyme, where some of the IDE is sequestered in peroxisomes. The fact that no significant difference was observed may suggest that the further increase in the cytosolic enzyme population was no longer rate-limiting for insulin degradation. Alternatively, the incremental increase in cytosolic IDE may have been too small to detect because of an already high proportion of cytosolic enzyme, underestimated in immunofluorescence experiments as a result of the diffuse nature of cytosolic staining. Finally, it may also result from retrafficking of the peroxisomal enzyme population to a location that is not involved in insulin degradation, such as the nucleus. Although insulin is internalized via receptor-mediated endocytosis, and can be degraded in endosomes or lysosomes (reviewed in Ref. 3), the hormone has been also detected in the cytoplasm and the nucleus (47– 49). Several studies suggest that a second and sometimes a third pathway may exist for insulin internalization, involving noncoated pits and fluid-phase endocytosis (50, 51). More recently, the hypothesis has been proposed that undegraded insulin could exit early endosomes and be translocated to the nucleus via the cytoplasm (48, 49). Degradation of insulin in the cytoplasm is blocked in a number of cells by metalloprotease inhibitors of the type that inhibits IDE (8 –10). Furthermore, specific anti-IDE antibodies were shown to block insulin degradation in cell cultures, supporting a role for IDE in insulin degradation (11, 14). Taken together, these data are consistent with a cytosolic pathway for insulin degradation by IDE. It is likely that IDE plays different roles in peroxisomes and in the cytosol. IDE is a member of a recently described family of metallopeptidases that share a HXXEH Zn21 binding motif in their catalytic domain. Interestingly, several members of this family have been implicated in proteolytic maturation processes, such as presequence processing of nuclear encoded mitochondrial proteins (52, 53), or prohormone maturation (23). IDE could thus perform a similar function in peroxisomes. Although most peroxisomal proteins are synthesized in their mature state, some exceptions have been shown to require proteolytic maturation (27, 28). In these cases, the absence of processing in mutant cells defective in peroxisome biogenesis suggests that the proteolytic processing events are performed by one or more proteases present, but still unidentified, in peroxisomes (28). Interestingly, the potential multifunctional role of IDE may extend to other members of its family. Indeed, two yeast homologs, Axl1p and Ste23, have recently been implicated in a-factor pheromone precursor processing (23). In addition, Axl1p appears to function as a morphogenetic determinant for axial bud selection. Furthermore, amino acid substitutions in the HXXEH motif caused defects in propheromone processing but failed to perturb bud site selection, suggesting

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that participation of Axl1p in axial budding does not require proteolysis. Because most of the proteins of this family of metallopeptidases are especially large compared with common proteases, this observation raises the possibility of a division of the protein into distinct functional domains. Although IDE and mitochondrial processing peptidase are quite divergent in terms of amino acid sequence, size and quaternary structure, mitochondrial processing peptidase b-subunit also contains the HXXEH consensus pentapeptide (52, 53). In addition to its involvement in presequence proteolytic maturation of nucleus-encoded mitochondrial proteins, this metallopeptidase has also been reported to be part of the respiratory chain bc1 complex in Neurospora crassa and plants, although the two functions appear clearly distinct in yeast and mammals. Because mutations that alter IDE’s peroxisomal targeting signal do not alter IDE’s ability to regulate cellular insulin degradation, it appears that this function does not require peroxisomal localization. This suggests that IDE may be a multifunctional protein and that other peroxisomal functions for IDE should continue to be sought. The diversity of biochemical reactions performed by peroxisomes leaves open a wide range of possible roles. References 1. Czech MP 1989 Signal transmission by the insulin-like growth factors. Cell 59:235–238 2. Rosen OM 1987 After insulin binds. Science 237:1452–1458 3. Duckworth WC 1988 Insulin degradation: mechanisms, products, and significance. Endocr Rev 9:319 –345 4. Levy JR, Olefsky JM 1986 Retroendocytosis of insulin in rat adipocytes. Endocrinology 119:572–579 5. Gehm BD, Kuo W-L, Perlman RK, Rosner MR 1993 Mutations in a zincbinding domain of human insulin-degrading enzyme eliminate catalytic activity but not insulin binding. J Biol Chem 268:7943–7948 6. Hamel FG, Posner BI, Bergeron JJM, Frank BH, Duckworth WC 1988 Isolation of insulin degradation products from endosomes derived from intact rat liver. J Biol Chem 263:6703– 6708 7. Duckworth WC, Hamel FG, Peavey DE, Liepnieks JJ, Ryan MP, Hermodson MA, Frank BH 1988 Degradation products of insulin generated by hepatocytes and insulin protease. J Biol Chem 263:1826 –1833 8. Kayalar C, Wong WT 1989 Metalloprotease inhibitors which block the differentiation of L6 myoblasts inhibit insulin degradation by the endogenous insulin-degrading enzyme. J Biol Chem 264:8928 – 8934 9. Kayalar C, Wong WT, Hendrickson L 1990 Differentiation of BC3H1 and primary skeletal muscle cells and the activity of their endogenous insulindegrading enzyme are inhibited by the same metalloendoprotease inhibitors. J Cell Biochem 44:137–151 10. Gehm BD, Rosner MR 1991 Regulation of insulin, epidermal growth factor and transforming growth factor alpha levels by growth factor degrading enzymes. Endocrinology 128:1603–1610 11. Shii K, Roth RA 1986 Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease. Proc Natl Acad Sci USA 83:4174 – 4151 12. Hari J, Shii K, Roth RA 1987 In vivo association of [125I]-insulin with a cytosolic insulin-degrading enzyme: detection by covalent cross-linking and immunoprecipitation with a monoclonal antibody. Endocrinology 120:829 – 831 13. Kuo W-L, Gehm BD, Rosner MR 1991 Regulation of insulin degradation: expression of an evolutionarily conserved insulin-degrading enzyme increases degradation by an intracellular pathway. Mol Endocrinol 5:1467–1476 14. Semple JW, Lang Y, Speck ER, Delovitch TL 1992 Processing and presentation of insulin. III. Insulin degrading enzyme: a neutral metalloendoproteinase that is non-homologous to classical endoproteinases mediates the processing of insulin epitopes for helper T cells. Int Immunol 4:1161–1167 15. Roth RA, Mesirow ML, Yokono K, Baba S 1984 Degradation of insulin-like growth factors I and II by a human insulin-degrading enzyme. Endocr Res 10:101–112 16. Garcia JV, Gehm BD, Rosner MR 1989 An evolutionarily conserved enzyme degrades transforming growth factor-alpha as well as insulin. J Cell Biol 109:1301–1307 17. Mu¨ller D, Schulze C, Baumeister H, Buck F, Richter D 1992 Rat insulindegrading enzyme: cleavage pattern of the natriuretic peptide hormones ANP,


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