J. Inher. Metab. Dis. 14 (1991) 152-164 (@SSIEM and Kluwer AcademicPublishers. Printed in the Netherlands

Acyl-CoA Oxidase, Peroxisomal Thiolase and Dihydroxyacetone Phosphate Acyltransferase: Aberrant Subcellular Localization in Zellweger Syndrome C. W. T. VAN ROERMUND 1, S. BRUL2, J. M. TAOER2, R. B. H. SCHUTGENS 1 and R. J. A. WANDERS 1. 1Department of Paediatrics, University Hospital Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands 2E. C. Slater Institute for Biochemical Research, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

Summary: We have studied the presence and subcetlular localization of peroxisomal 3-oxoacylcoenzyme A thiolase, acylcoenzyme A oxidase and acylCoA: dihydroxyacetonephosphate acyltransferase (DHAPAT) in fibroblasts from control subjects and patients with an inherited deficiency of peroxisomes (Zellweger syndrome), using immunofluorescence spectroscopy and density gradient centrifugation techniques. The results show that Zellweger cells contain unprocessed thiolase and unprocessed acyl-CoA oxidase which are associated with structures containing a peroxisomal integral membrane protein of 69 kDa and having a density much lower than that of normal peroxisomes. The residual DHAPAT activity present in Zellweger cells is also contained in these structures. We conclude that these structures represent defectively assembled peroxisomes which may still be capable of importing some peroxisomal proteins.

INTRODUCTION Peroxisomes are subcellular organelles which are present in most, if not all, mammalian cells, other than mature erythrocytes. Studies during the last few years have shown that peroxisomes play an indispensable r61e in mammalian metabolism. These organelles are involved in the synthesis of ether-phospholipids and bile acids, in the fi-oxidation of fatty acids, prostaglandins and other compounds, in the breakdown of pipecolate and glyoxylate and in other metabolic pathways (see Wanders et aL, 1988 and Lazarow and Moser, t989). The importance of peroxisomes in man is stressed by the existence of a group of inherited diseases, the peroxisomal disorders, in which there is an impairment in one or more peroxisomal functions. The prototype of this group of disorders is the cerebro-hepato renal syndrome of

MS received 17.10.90 Accepted 26.11.90 *Correspondence 152

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Zellweger, in which morphologically distinguishable peroxisomes are deficient, as first shown by Goldfischer and colleagues (1973). Peroxisomes are also deficient in patients suffering from the neonatal form of adrenoleukodystrophy and the infantile form of Refsum disease. These three disorders are collectively called disorders of peroxisome biogenesis. Although it was long thought that peroxisomes arise by budding from the endoplasmic reticulum, it is now clear that peroxisomes originate by division, or fission, of pre-existing ones (see Fujiki and Lazarow, 1985; Borst, 1986 and 1989 for reviews). Another, now generally accepted, principle of microbody biogenesis is that peroxisomal matrix proteins and membrane proteins are encoded for by nuclear genes and translated on free polyribosomes, as first shown for urate oxidase and catalase by Goldman and Blobel (1978) and Robbi and Lazarow (1978). In the last few years considerable information has become available on the targeting signals which direct peroxisomal proteins to their final subcellular destination, the peroxisome. Gould and colleagues (1987, 1989) found that the information for the targeting of luciferase was contained in the carboxy terminal three amino acids of the protein, serine-lysine-leucine (SKL) (Gould et al., 1989). When this tripeptide was fused onto a bacterial protein {chloramphenicol acetyltransferase) at the carboxy terminus, the fusion protein was indeed directed to peroxisomes (Gould et al., 1989). Further studies revealed that a limited number of substitutions can be made within the SKL-sequence without eliminating its activity (Gould et al., 1989). Additional evidence emphasizing the importance of the carboxy-terminal SKL-sequence in peroxisome targeting comes from in vitro import experiments by Miyazawa and colleagues (1989) using rat acylCoA oxidase. The finding that carboxy terminal SKL-sequences (or one of its variants) occur in a number of different peroxisomal proteins has led to the suggestion that this constitutes at least one peroxisomal targeting signal. Studies in fibroblasts from Zellweger patients have shown that peroxisomal enzymes such as acyl-CoA oxidase and thiolase are synthesized normally but degraded rapidly in the absence of peroxisomes (Schram et al., 1986). This finding raised the question of whether the defect was in the assembly of the peroxisomal membrane or in the import machinery for peroxisomal proteins. This was first studied by Lazarow and colleagues (1986), who reported that the 22 kDa integral membrane protein was not deficient in the liver of a Zellweger patient. Subsequent studies by a number of investigators (Lazarow et al., 1986; Suzuki et al., 1987, 1989; Small et al., 1988; Wiemer et al., 1989) have shown that peroxisomal membrane proteins are in general not completely deficient but that there are quantitative differences in the amounts present in material from different patients. Studies by Santos and colleagues (1988a, 1988b) and Wiemer and colleagues (1989) have clearly shown that these membrane proteins are contained within closed structures which are in general much larger in size than normal peroxisomes, and are apparently defective in protein import. As an extension of such studies we have now investigated the subcetlular localization of the peroxisomal enzymes 3-oxoacyl-CoA thiolase, acyl-CoA:dihydroxyacetonephosphate acyltransferase and acyl-CoA oxidase, as well as that of the 69 kDa peroxisomal integral membrane protein, in control and Zellweger fibroblasts using different techniques. The results are described in this paper. J. Inher. Metab. Dis. 14 (1991)

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MATERIALS AND METHODS

Cell lines and culture conditions: Cultured skin fibroblasts were obtained from patients with the cerebro-hepato-renal (Zellweger) syndrome and from controls, and cultured as previously described (Wanders et al., 1987). Cell lines from Zeltweger patients were biochemicatly characterized by demonstrating a deficiency of dihydroxyacetonephosphate acyltransferase, particle-bound catalase and hexacosanoate (C26: 13) fl-oxidation, strongly reduced ptasmalogen levels, impaired de novo synthesis of plasmalogens, accumulation of very long chain fatty acids and an absence of punctate fluorescence upon staining of fixed cells with anti-(catalase) antibodies (see Wanders et at., 1988 for details). Preparation of antibodies: Anti-(catalase) antibodies were raised as previously described (Tager et al., 1985). Antisera against acyl-CoA oxidase and peroxisomal 3oxoacyl-CoA thiolase were prepared as described (Osumi et al., 1980; Miyazawa et al., 1981) using the enzyme proteins purified from rat liver. Anti-(69 kDa peroxisomal membrane protein) antibodies were prepared as previously described (Hashimoto et al., 1986). As shown previously (Tager et al., 1985; Wiemer et at., 1989), all antisera crossreact with the corresponding human enzyme proteins. Immunofluorescence microscopy: The procedure used for immunofluorescence microscopy was as previously described (Wiemer et al., 1989). In short, cells were seeded and grown on glass coverslips, washed twice with phosphate buffered saline (PBS) at near confluency, and subsequently fixed and permeabilized with a freshly prepared solution containing 2% (mass/vol) paraformaldehyde and 0.1% (by vol) Triton X-100 in PBS. Subsequently, the cells were rinsed with 0.1% (by vol) Triton X-100 in PBS and free aldehyde groups quenched by incubating the cells for 10min in a medium containing 0.1 mol/L NH4C1 in PBS. Finally, the cells were washed twice with PBS. Primary antibody incubations were performed in PBS containing 10mg/ml albumin for 30 rain at room temperature. The cells were then incubated successively with a biotin-conjugated anti-(rabbit IgG) serum followed by streptavidin labelled with fluorescein isothiocyanate (see Wiemer et at., 1989 for details). The coverslips were mounted on microscope slides in carbonate-buffered glycerol containing p-phenylenediamine, sealed with nail polish and stored at - 20°C. Fluorescence was viewed with an Olympus fluorescence microscope equipped with a camera and Orthomat E control unit, using a filter with excitation wavelength of 450-490 nm. Fractionation offibrobtasts: Cells were grown in 150 on3. 2 culture flasks (7-9 flasks per experiment) in Ham F10 medium supplemented with 15% (by vol) fetal calf serum, 16mmol/L NaHCO3, penicillin (250mU/ml), and streptomycin (250#g/ml). Three days after confluency cells were harvested by gentle trypsinization as previously described (Wanders et al., 1987). The cells were collected by centrifugation and washed twice. Finally, the cells were washed in ice-cold homogenization buffer (0.25 mmol/L sucrose, 0.1 mmol/L EDTA, 1 mmol/L Tris-HC1, pH 7.8) plus protease inhibitors (leupeptin, pepstatin and phenylmethylsulphonyl chloride (PMSF)) and J. lnher. Metab. Dis. 14 (1991)

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postnuclear supernatants were prepared as previously described (Wanders et al., 1990). Subsequently, equilibrium density gradient centrifugation was performed by layering 5 ml of the postnuclear supernatant (containing about 25 mg of protein) on top of a preformed gradient of Nycodenz (10-> 35 % (mass/vol)), prepared by stepwise layering of Nycodenz solutions of increasing density. All Nycodenz solutions were prepared in homogenization buffer. Centrifugation was carried out in an MSEEurope 24M centrifuge, equipped with a vertical rotor, for 90min at 19000rpm at 4°C. Enzyme determinations: Glutamate dehydrogenase, lactate dehydrogenase, catalase and dihydroxyacetonephosphate acyltransferase were measured as previously described (Wanders et al., 1984; Schutgens et al., 1986). The activity of acyl-CoA oxidase was determined radiochemically using a newly devised method. The method is based on the formation of [14C]acetyl-CoA from [14C]palmitoyl-CoA as catalysed by acyt-CoA oxidase plus added crotonase, 3-hydroxyacyl-CoA dehydrogenase and thiolase to ensure degradation of the enoyl-CoA formed to acetyl-CoA. Activity was measured in the following medium: 100 mmol/L morpholinopropanesulphonic acidNaOH, 100#mol/L FAD, l mmol/L NAD +, l mmol/L eoenzyme A, 2mmol/L pyruvate, 0.75 U/ml lactate dehydrogenase, 0.75 U/ml 3-hydroxyacyl-CoA dehydrogenase, 0.35 U/ml crotonase, 3.6 mU/ml thiolase and 100 #mol/L palmitoyl-CoA. The final pH was 8.5. Reactions were started by addition of sample in a final volume of 0.2 ml. After 30 min at 37°C, reactions were terminated by addition of perchloric acid (final concentrations 1 mol/L). The [14C]acetyl-CoA produced was separated from the [14C]palmitoyl-CoA as previously described (Wanders et aI., 1990). Immunobtot analysis: 1 mt portions of each of the fractions obtained after density gradient centrifugation were first treated with ice-cold trichloroacetic acid (TCA) to a final concentration of 10% (mass/vol). Following centrifugation, precipitates were directly dissolved in 300 #1 of sample buffer containing 20 mmol/L Tris-HC 1, pH 8.0, 2 mmot/L EDTA, 2% SDS, 20% (by vol) glycerol, 1.42 mmol/L/~-mercaptoethanol and 0.005% (mass/vol) bromophenol blue. Samples were subsequently subjected to etectrophoresis on polyacrylamide gels in the presence of SDS as previously described (Tager et at., 1985). Immunoblot analysis was performed as described (Tager et al., 1985). Antigen-antibody complexes were visualized by incubation with goat antirabbit Ig conjugated to peroxidase, followed by colour development using tetramethylbenzidine.

RESULTS Immunofluorescence studies in cultured fibroblasts from controls and Zellweger patients

It was found earlier that peroxisomal 3-oxoacyl-CoA thiolase is present in total homogenates of liver and/or fibroblasts from Zellweger patients in the 44kDa precursor form, the 41kDa mature form being absent (Tager et al., 1985; see also J. Inher. Metab. Dis. 14 (1991)

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Suzuki et al., 1986; Chen et aI., 1987; Shimozawa et al., 1988; Guerroui et aI., 1989; Wiemer et al., 1989). Furthermore, the 70kDa component of acyl-CoA oxidase is present in Zellweger patients, in contrast to the 50kDa and 20kDa components which are deficient (Tager et aI., 1985; Suzuki et aI., 1986; Chen et al., 1987; Guerroui et al., 1989; Wiemer et al., 1989). This led us to investigate the subcellular localization of these enzyme proteins in fibroblasts from Zellweger patients. This was first done by carrying out immunofluorescence studies using antibodies raised against rat liver 3-oxoacyl-CoA thiolase and acyl-CoA oxidase, respectively. Visualization occurred by an indirect immunofluorescence labelling technique employing a biotin streptavidin signal enhancement step as previously described (Wiemer et at., 1989). When control fibroblasts were incubated with specific polyclonal antibodies directed against catalase (A), the 69 kDa P M P (B), acyl-CoA oxidase (C) and peroxisomal thiolase (D), a punctate fluorescence staining pattern was observed in all cases (Figure tA-D). In the case of Zellweger fibroblasts, a diffuse staining pattern was observed in the case of catalase (Figure 1E), in line with previous results (Santos et al., 1988b; Wiemer et al., 1989). When Zellweger fibroblasts were stained for the 69 kDa PMP, the fluorescence pattern was punctate again, although the spots appeared somewhat larger as compared to control fibroblasts (Figure 1F). When Zellweger fibroblasts were probed with antiserum against acyl-CoA oxidase and peroxisomal thiolase (Figure 1, G and H respectively), a punctate fluorescence pattern was observed reminiscent of the picture observed with the anti-(69kDa PMP) antiserum but completely different from that seen in the case of catatase. A similar pattern of punctate fluorescence was recently reported by Balfe and colleagues (1990) in the case of peroxisomal thiolase. A problem with the use of immunofluorescence for establishing the subcellular localization of a protein is that the antisera used must be monospecific for the protein in question. Although this has been verified for the antisera we used by means of immunoelectronmicroscopy (Espeel et al., 1990), we have carried out additional experiments using density gradient centrifugation. Subcellular fractionation studies

To obtain further information on the intracellular localization of peroxisomal 3oxoacyl-CoA thiolase and acyl-CoA oxidase in Zellweger fibroblasts, postnuclear supernatants of fibroblast homogenates were subjected to Nycodenz density gradient centrifugation as described in the Materials and Methods section. Glutamate dehydrogenase was measured in the fractions as a mitochondrial marker enzyme, lactate dehydrogenase as a cytosolic marker and catalase and dihydroxyacetonephosphate acyttransferase (DHAPAT) as peroxisomal markers. When assayed at pH 5.5 in the presence of glycerol-3-phosphate, DHAPAT is the marker of choice for peroxisomes (De Clerq et al., 1984). The left hand panel of Figure 2 shows that in a gradient of control cells, peroxisomes are well resolved from mitochondria and cytosol, peaking in fractions 2, 4 and 8 respectively. Whereas DHAPAT shows a unimodal distribution pattern, a bimodal pattern was observed in the case of catalase which is due to breakage of peroxisomes on homogenization. Figure 2 further shows that acyt-CoA oxidase also peaks in fraction 2 (Figure 2C, left hand panel). J. lnher. Metab. Dis. t4 (1991)

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Figure 1 Presence and subcellular localization of catalase, the 69 kDa peroxisomal integral membrane protein, acyl-CoA oxidase and peroxisomaI thiolase in fibroblasts from a control subject and a patient with the cerebro-hepato-renal syndrome of Zellweger. Fibroblasts were grown to near confluency, fixed in 2% (w/v) paraformaldehyde containing 0.1% (w/v) Triton X-100 and probed with specific antibodies directed against catalase (A, E), the 69kDa peroxisomal membrane protein (B, F), acyl-CoA oxidase (C, G) and peroxisomal thiolase (D, H) followed by biotinylated donkey anti-(rabbit Ig), and finally streptavidin labelled with FITC. Control cells: A D; Zellweger cells: E-H.

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On fractionation of Zellweger fibroblasts (Figure 2, right hand panel) the distribution patterns for glutamate dehydrogenase (D) and lactate dehydrogena~se (E) were similar to those seen in control fibroblasts. Markedly different distribution patterns were seen in the cases of catalase, DHAPAT and acyl-CoA oxidase (A, B and C of Figure 2, right panel). Catalase was found on top of the gradient, the highest activity being observed in fraction 8. This profile of activity corresponds to that of lactate dehydrogenase (E), which is in line with the known cytosolic localization of catalase in Zellweger fibroblasts. When the activity of DHAPAT was measured, however, a profile different from that of catalase was found: indeed, activity was highest in fraction 5. An identical profile of activity was found when acyl-CoA oxidase activity was measured (Figure 2C, right hand panel).

Immunoblot analysis of the different fractions obtained by density gradient centrifugation We subsequently performed immunoblot analysis of the fractions obtained by density gradient centrifugation (Figure 3). In control fibroblasts, the 50kDa and 20kDa components of acyl-CoA oxidase were found to be most abundant in fraction 2 which is in line with the results of activity measurements of acyl-CoA oxidase (see Figure 2, right panel). Also, peroxisomal thiolase and the 69 kDa PMP intensities peaked in fraction 2 in control fibroblasts. In Zellweger fibroblasts, however, no crossreactive material was found in fraction 2 using any of the antibodies. The intensity of the 72 kDa component of acyl-CoA oxidase is highest in fraction 6, whereas for the 69 kDa P M P the highest intensity is seen in fraction 5, a faint band being detectable in fraction 8 containing the bulk of the cytosolic proteins. Finally, 3-oxoacyl-CoA thiolase was found to peak in fraction 5.

DISCUSSION Earlier studies have shown that whereas several peroxisomal enzyme activities are deficient in Zellweger patients, other enzyme activities are normally active. The peroxisomal matrix enzymes catalase, D-amino acid oxidase, L-c~-hydroxyacid oxidase A and alanine glyoxylate aminotransferase are normally active in Zellweger cells, but are located in the cytoplasmic compartment (see Lazarow and Moser, 1989 for review). As first shown for the 22 kDa integral membrane protein by Lazarow and colleagues (1986), it has now become clear that various membrane proteins are also present in Zellweger cells (Lazarow et al., 1986; Suzuki et al., 1987, 1989; Small et al., 1988; Santos et at., 1988a, 1988b; Wiemer et al., t989). Santos and colleagues (1988a, 1988b) (see also Wiemer et al., 1989) have shown that these membrane proteins are contained in vesicles which exhibit a density lower than that of normal peroxisomes, as demonstrated by subcellular gradient centrifugation experiments in cultured skin fibroblasts. The results described in this paper confirm that the 69 kDa PMP is indeed localized in a fraction with a density lower than that of normal peroxisomes (Figure 3). Furthermore, our results suggest that these structures do not only contain the 69 kDa membrane protein but also unprocessed thiolase (which is J. Inher. Metab. Dis. 14 (1991)

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Figure 2 Density gradient centrifugation of control and Zellweger fibroblasts. Fibroblasts from a control subject and a Zellweger patient were fractioned on Nycodenz gradients as described in the Materials and Methods section. This was followed by measurement of catalase (A), DHAPAT (B), acyl-CoA oxidase (C), glutamate dehydrogenase (D), lactate dehydrogenase (E) and protein (F) in the different fractions. Left hand panel: control cells; right hand panel: Zellweger cells.

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in agreement with recent results published by Balfe and colleagues (1990)), unprocessed acyl-CoA oxidase and dihydroxyacetonephosphate acyltransferase activity. According to the results in Figures 2 and 3, the precursor form of acyl-CoA oxidase is not only immunologicatly detectable in the 69 kDa containing structures but is also enzymatically active (see Figure 2C, right panel). Future studies wilt have to reveal in what molecular form DHAPAT is present in peroxisomal ghosts in Zellweger patients. The finding that Zellweger fibroblasts contain low density particles containing not only the 69 kDa integral membrane protein but also unprocessed thiolase and acytCoA oxidase as well as dihydroxyacetonephosphate acyltransferase is intriguing, and suggests that the assembly of peroxisomes is defective in these cells, without affecting each enzyme protein to the same extent. Furthermore, it is clear that the enzyme proteins contained in these structures are not only membrane-bound enzyme proteins, since acyl-CoA oxidase and 3-oxoacyl-CoA thiolase are also present in these defectively assembled organelles, albeit in their unprocessed form. Immunoelectronmicroscopy studies by Yokota and colleagues (1987) have shown that these enzyme proteins are predominantly localized in the soluble interior of peroxisomes, at least in rat liver. Future immunoelectronmicroscopic studies will have to be done to unravel the exact localization of unprocessed thiolase and acyl-CoA oxidase in the aberrant peroxisome structures in Zellweger cells. An intriguing question is how these results can be intrepreted within the concept of peroxisome biogenesis. Since the targeting information which directs proteins to the peroxisome is contained within the polypeptide sequence itself, it is clear that the primary defect in Zellweger syndrome is either at the level of the recognition of peroxisomal proteins, e.g. by putative receptors located at the peroxisomal membrane, or at the level of the import process itself. Studies by Gould and colleagues (1987, 1989) have shown that a serine-lysine~-leucine (SKL) tripeptide sequence located at the carboxy terminal end of certain peroxisomal proteins is able to direct them to peroxisomes. Carboxy terminal addition of a single amino acid onto the SKLsequence abolishes peroxisome localization. On the other hand, an internal SKLsequence in the peroxisomal matrix protein catalase appears to be active as a peroxisome targeting signal (Gould et al., 1988). Apparently, the peroxisome targeting sequence must be exposed properly in order to allow interaction with proteins of the import machinery. Importantly, sequence comparison of peroxisomal proteins has shown that many contain a SKL or SKL-variant. This applies for D-amino acid oxidase (from pig liver), acyl-CoA oxidase (from rat liver), bifunctional protein (from rat liver), firefly luciferase and urate oxidase (from soyabean) (Gould et aL, 1988). Peroxisomal thiolase, however, both from rat liver (Hijikata et al., 1987) and from human liver (Bout et aI., 1988), lacks a carboxy terminal SKL-sequence. Very recently, Kamijo and colleagues (1990) sequenced the 69 kDa integral membrane protein from rat liver, and the protein was also found to lack a carboxy terminal SKL sequence. These findings raise the possibility that the SKL-sequence is not the only peroxisome targeting signal. Using antibodies directed against the peroxisomal targeting SKL signal, Gould and colleagues (1990) recently obtained evidence suggesting that many but not all peroxisomal enzyme proteins contain the SKL sequence, whereas other d. Inher. Metab. Dis. 14 (1991)

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proteins do not, which suggests that they are targeted to the peroxisome via another mechanism. Following this line of reasoning, it could be that the targeting of all those enzyme proteins which have an SKL targeting signal is defective in Zellweger syndrome e.g. due to a non-functional SKL receptor. This would explain why peroxisomal enzyme proteins such as catalase and D-amino acid oxidase are localized in the cytosolic compartment in Zellweger cells, whereas the 69kDa integral membrane protein and 3-oxoacyl-CoA thiolase are present in membranous structures. The finding that acyl-CoA oxidase is also associated with these structures, however, is not easy to reconcile with this suggestion since acyl-CoA oxidase, at least from rat liver, contains a carboxy terminal SKL sequence. One possibility which cannot be excluded at this time is that acyl-CoA oxidase is not localized within these structures, but instead is only associated with them. Immunoelectronmicroscopic studies are underway to resolve this question.

ACKNOWLEDGEMENTS This work was supported by grants from The Princess Beatrix Fund and the Netherlands Organization for Scientific Research (NWO) under the auspices of the Netherlands Foundation for Medical and Health Research (MEDIGON). Mariska van Doom is gratefully acknowledged for expert preparation of the manuscript and Prof. T. Hashimoto for providing antibodies. We gratefully thank Judith Heikoop, Rob Ofman, Anita Schelen, Eric Wiemer and Ernst Wolvetang for their help.

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