Biogenesis of peroxisomes. Topogenesis of the peroxisomal membrane and matrix proteins

Genetic and proteomic approaches have led to the identification of 32 proteins, collectively called peroxins, which are required for the biogenesis of peroxisomes. Some are responsible for the division and inheritance of peroxisomes; however, most peroxins have been implicated in the topogenesis of peroxisomal proteins. Peroxisomal membrane and matrix proteins are synthesized on free ribosomes in the cytosol and are imported post-translationally into pre-existing organelles (Lazarow PB & Fujiki Y (1985) Annu Rev Cell Biol1, 489-530). Progress has been made in the elucidation of how these proteins are targeted to the organelle. In addition, the understanding of the composition of the peroxisomal import apparatus and the order of events taking place during the cascade of peroxisomal protein import has increased significantly. However, our knowledge on the basic principles of peroxisomal membrane protein insertion or translocation of peroxisomal matrix proteins across the peroxisomal membrane is rather limited. The latter is of particular interest as the peroxisomal import machinery accommodates folded, even oligomeric, proteins, which distinguishes this apparatus from the well characterized translocons of other organelles. Furthermore, the origin of the peroxisomal membrane is still enigmatic. Recent observations suggest the existence of two classes of peroxisomal membrane proteins. Newly synthesized class I proteins are directly targeted to and inserted into the peroxisomal membrane, while class II proteins reach their final destination via the endoplasmic reticulum or a subcompartment thereof, which would be in accord with the idea that the peroxisomal membrane might be derived from the endoplasmic reticulum.     

In vitro insertion of the 22-kD peroxisomal membrane protein into isolated rat liver peroxisomes

The membrane insertion of the 22-kD integral peroxisomal membrane protein (PMP 22) was studied in a system in which peroxisomes isolated from rat liver were incubated with the [35S]methionine-labeled in vitro translation product of PMP 22 mRNA. Membrane insertion of PMP 22 was demonstrated by protease treatment of peroxisomes in the absence and presence of detergent. Approximately 35% of total in vitro translated PMP 22 became protease resistant after a 1-h incubation at 26 degrees C. Import was dependent on time and temperature, did not require ATP or GTP and was not inhibited by N-ethylmaleimide treatment of neither the soluble components of the translation mixture nor of the isolated peroxisomes. In contrast to these results it was recently shown that the import of the peroxisomal marker, firefly luciferase, into peroxisomes of permeabilized cells was dependent on ATP hydrolysis and was blocked by N-ethylmaleimide pretreatment of the cytosol-depleted cells (Rapp et al., 1993; Wendland and Subramani, 1993). Therefore, the present data suggest that insertion of PMP 22 into the peroxisomal membrane and translocation of firefly luciferase into peroxisomes follow distinct mechanisms. At low temperature binding of PMP 22 to the peroxisomal membrane was not influenced whereas insertion was strongly inhibited. Pretreatment of peroxisomes with subtilisin reduced binding to a low level and completely abolished insertion. Therefore it is suggested that binding is prerequisite to insertion and that insertion may be mediated by a proteinaceous receptor.     

Effects of antilipolytic agents on rat liver peroxisomes and peroxisomal oxidative activities

The mechanisms involved in the inhibitory effects of antilipolytic agents on rat liver peroxisomal fatty acid oxidative activity have been explored. Treatment of fasting rats with antilipolytic drugs (either 3,5-dimethylpyrazole (12 mg/kg body weight) or Acipimox (25 mg/kg body weight)) resulted in a decrease in free fatty acid and glucose plasma levels within 5–10 and in a significant increase in the plasma glucagon to insulin ratio within 15. Changes in the fatty acid oxidative activity appeared with a 2.5–3 h delay and were then very rapid (a 30–40% decrease in the activity occured in additional 2 h). Many peroxisomal enzyme activities (including non-β-oxidative activities such as uricase and D-amino acid oxidase) exhibited similar changes with the same delay. Simultaneously with the enzyme changes, at the electron microscope level many autophagic vacuoles were detected in the liver cells, often containing peroxisomal structures. Glutamine, an inhibitor of proteolysis in vivo, prevented the decrease in enzyme activities. It was concluded that the decrease in peroxisomal enzyme activities may be the consequence of enhanced peroxisome degradation due to the stimulation of autophagic processes in liver cells.     

Purification of membrane polypeptides of rat liver peroxisomes

Peroxisomes were obtained by sucrose density gradient centrifugation from the livers of di(2-ethylhexyl)phthalate-fed rats, and the membranes were prepared by carbonate extraction (Fujiki, Y., Fowler, S., Shio, H., Hubbard, A.L., & Lazarow, P.B. (1982) J. Cell Biol. 93, 103-110). The integrated membrane polypeptides were solubilized with sodium dodecyl sulfate, and purified by repeated polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Separation of 70 and 68 kDa polypeptides was not attempted in the present study because of their close migration in polyacrylamide gel electrophoresis. Other polypeptides with apparent molecular masses of 41, 27, 26, and 22 kDa were purified to near homogeneity. Antibodies were raised against these purified preparations. The 68 kDa polypeptide is suggested to be produced by the proteolytic modification of 70 kDa polypeptide, since the former increased concomitantly with decrease of the latter when the liver homogenate was incubated, and this change was prevented in the presence of leupeptin during the incubation. The 41 kDa polypeptide was a minor component. The 70 and 68 kDa polypeptides and 41 kDa polypeptide and their antibodies were cross-reactive, but the relation of these polypeptides was not clear. The 27 and 26 kDa polypeptides seemed to be another species of membrane polypeptides, although the relationship of these two polypeptides remains to be clarified. The 22 kDa polypeptide is not related to other membrane polypeptides. The results of immunoblot analysis of subcellular fractions of the liver and an electron microscopic immunocytochemical study to locate the antigenic sites with protein A-gold complex suggest that all of these polypeptides are localized on peroxisomal membranes. On proliferation of rat liver peroxisomes by administration of di(2-ethylhexyl)phthalate, a peroxisome proliferator, all of these polypeptides were markedly increased.     

Biosynthesis of membrane polypeptides of rat liver peroxisomes

The biosynthesis of three major peroxisomal membrane polypeptides of rat liver was investigated. Total hepatic RNA extracted by the guanidinium/CsCl method from three control and three di(2-ethylhexyl)phthalate (a peroxisomal proliferator)-fed rats was translated in vitro in a rabbit reticulocyte lysate protein-synthesizing system. Translation products were immunoprecipitated by the antibodies against peroxisomal membrane polypeptides, subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and analyzed by fluorography. The in vitro translation products of 70, 26, and 22 kDa peroxisomal membrane polypeptides were apparently of the same size as the respective mature polypeptides. The ratio of translatable mRNA levels for the 70, 26, and 22 kDa polypeptides in di(2-ethylhexyl)phthalate-fed rats to those in control rats were 5.4, 11.4, and 2.7, respectively. The synthesis of these three polypeptides with the free polysome fraction from di(2-ethylhexyl)phthalate-fed rats was more active than that with the membrane-bound polysome fraction, whereas the synthesis of albumin with the free polysome fraction was 27% of that with the membrane-bound polysome fraction. These results indicate that the peroxisomal major membrane polypeptides are synthesized on free polysomes and transported to peroxisomal membrane without any apparent proteolytic processing, and that the induction of these polypeptides by administration of a peroxisomal proliferator corresponds well to the induction of the peroxisomal beta-oxidation enzymes. The data also support the idea that peroxisomes are organized from pre-existing peroxisomes.     

Biogenesis of peroxisomes: sequential biosynthesis of the membrane and matrix proteins in the course of hepatic regeneration

The biogenesis of peroxisomes was investigated in the model of regenerating rat liver after partial hepatectomy (PH), using analytical differential centrifugation in combination with immunoblotting and in vivo pulse labeling as well as immunoelectron microscopy. The total activity of catalase decreased sharply after PH, returning gradually over several days to normal levels. In the 16 to 32-h period the enzyme activity started to increase first in the heavy mitochondrial fraction, shifting at 28 h to the crude peroxisomal and at 32 h to the microsomal fraction, suggesting de novo formation of peroxisomes by budding or fragmentation from larger aggregates. Whereas most peroxisomal matrix proteins were reduced during the 16 to 32-h period after PH, the 26 and 70 kDa peroxisomal membrane proteins were increased. Moreover, in vivo pulse labeling studies with radioactive leucine showed significantly higher levels of specific activity in the peroxisomal membrane than in the matrix subfractions at 16 h with increasing labeling of the matrix at 32 h after PH. These findings suggest that de novo formation of peroxisomes in regenerating rat liver is initiated by the synthesis of membrane proteins and is followed by that of the matrix components.     

The role of 15-lipoxygenase in disruption of the peroxisomal membrane and in programmed degradation of peroxisomes in normal rat liver.

Our earlier electron microscopic observations revealed that prolonged exposure of glutaraldehyde-fixed rat liver sections to buffer solutions induced focal membrane disruptions of peroxisomes with catalase diffusion as shown cytochemically. Recently, it was suggested that 15-lipoxygenase (15-LOX) might be involved in natural degradation of membrane-bound organelles in reticulocytes by integrating into and permeabilizing the organelle membranes, leading to the release of matrix proteins. We have now investigated the localization of 15-LOX and its role in degradation of peroxisomal membranes in rat liver. Aldehyde-fixed liver slices were incubated in a medium that conserved the 15-LOX activity, consisting of 50 mM HEPES-KOH buffer (pH 7.4), 5 mM mercaptoethanol, 1 mM MgCl(2), 15 mM NaN(3), and 0.2 M sucrose, in presence or absence of 0.5-0.05 mM propyl gallate or esculetin, two inhibitors of 15-LOX. The exposure of aldehyde-fixed liver sections to this medium induced focal disruptions of peroxisome membranes and catalase diffusion around some but not all peroxisomes. This was significantly reduced by both 15-LOX inhibitors, propyl gallate and esculetin, with the latter being more effective. Double immunofluorescent staining for 15-LOX and catalase revealed that 15-LOX was co-localized with catalase in some but not all peroxisomes in rat hepatocytes. By postembedding immunoelectron microscopy, gold labeling was localized on membranes of some peroxisomes. These observations suggest that 15-LOX is involved in degradation of peroxisomal membranes and might have a physiological role in programmed degradation and turnover of peroxisomes in hepatocytes. (J Histochem Cytochem 49:613-621, 2001)     

Identification of a catalase-negative sub-population of peroxisomes induced in mouse liver by clofibrate.

The peroxisomal compartment in mouse liver was investigated using rate sedimentation of liver subfractions on sucrose density gradients. Treatment of mice with clofibrate, a hypolipidemic agent and peroxisome proliferator, resulted in the formation of small particles which were devoid of catalase and urate oxidase, but which were identified as peroxisomal on the basis of content of the clofibrate-induced peroxisomal beta-oxidation enzymes (fatty acyl-CoA oxidase, hydratase/dehydrogenase bifunctional protein, and thiolase) and the 68 kDa peroxisomal integral membrane protein. Immunoelectron microscopy confirmed the membrane-bound organellar nature and enzyme composition of these particles. These particles were absent in normal mice, and were increased to a maximal level within 2 days of clofibrate treatment. These data have been taken as indicative of a role of these particles in the mechanism of drug-induced peroxisome proliferation.     

Enhancement of long-chain acyl-CoA hydrolase activity in peroxisomes and mitochondria of rat liver by peroxisomal proliferators

The present study has confirmed previous findings of long-chain acyl-CoA hydrolase activities in the mitochondrial and microsomal fractions of the normal rat liver. In addition, experimental evidence is presented in support of a peroxisomal localization of long-chain acyl-CoA hydrolase activity. (a) Analytical differential centrifugation of homogenates from normal rat liver revealed that this activity (using palmitoyl-CoA as the substrate) was also present in a population of particles with an average sedimentation coefficient of 6740 S, characteristic of peroxisomal marker enzymes. (b) The subcellular distribution of the hydrolase activity was greatly affected by administration of the peroxisomal proliferators clofibrate and tiadenol. The specific activity was enhanced in the mitochondrial fraction and in a population of particles with an average sedimentation coefficient of 4400 S, characteristic of peroxisomal marker enzymes. Three populations of particles containing lysosomal marker enzymes were found by analytical differential centrifugation, both in normal and clofibrate-treated rats. Our data do not support the proposal that palmitoyl-CoA hydrolase and acid phosphatase belong to the same subcellular particles. In livers from rats treated with peroxisomal proliferators, the specific activity of palmitoyl-CoA hydrolase was also enhanced in the particle-free supernatant. Evidence is presented that this activity at least in part, is related to the peroxisomal proliferation.     

Influence of subtotal hepatectomy on peroxisomes and peroxisomal enzymes of rat liver and isolated liver cell fractions.

The activities of peroxisomal enzymes of rat liver were followed 1 to 10 days after subtotal (60-70%) hepatectomy in homogenates prepared from regenerating livers and in cell fractions isolated from them. Catalase activity was found to be depressed in the total liver homogenate (H) as well as in the mitochondrial (M) and soluble (S) fractions, while it did not change appreciably in the microsomal (Mc) and lysosomal (L) fractions. Alpha-hydroxyacid oxidase behaved in a similar fashion. In contrast to these enzymes, urate oxidase activity remained unchanged in H, whereas it was decreased in M and increased in L and Mc during the first 5 days after operation. These results agree well with the assumption that microbody proliferation is initiated by the fragmentation of large peroxisomes. The different relations of peroxisomal enzyme activities during regeneration time are discussed with respect to the possible existence of various kinds of peroxisomes with different enzyme equipments and with different turnover rates. Biochemical examinations ions were paralleled to morphological and histochemical studies. An early increase in number of peroxisomes was found to occur during the first day after partial hepatectomy, which is accompanied by decrease in particle size. During the first mitotic wave (24-36 hrs post op.) the number of peroxisomes per cell was reduced to about the half. After this time number and size of the particles began to increase. Positive staining of ribosomes was frequently observed in the vicinity of peroxisomes after the application of the cytochemical catalase reaction (alkaline diaminobenzidine medium). This phenomenon is interpreted to represent rather a diffusion artifact than the cytochemical identification of newly synthesized catalase.     

Major ATPases on clofibrate-induced rat liver peroxisomes are not associated with 70 kDa peroxisomal membrane protein (PMP70).

We previously reported that novel Mg(2+)-ATPases were induced in rat liver peroxisomes by clofibrate administration and that these activities consisted of at least two types of enzymes, N-ethylmaleimide (NEM)-sensitive and -resistant. Here we present evidence that neither of these major peroxisomal ATPases is associated with the 70-kDa peroxisomal membrane protein (PMP70), because: (i) proteinase K treatment of peroxisomes resulted in inactivation of only NEM-sensitive ATPase, whereas disappeared PMP70 completely; (ii) NEM-sensitive ATPase activity was barely immunoprecipitated with anti-PMP70 IgG; (iii) the solubilized ATPases behaved differently from PMP70 on native PAGE; and finally (iv), the major peroxisomal ATPases were separated from PMP70 on gel filtration chromatography.     

Nucleic acid synthesis in proliferating peroxisomes of rat liver as revealed by electron microscopical radioautography

Summary Thirty albino rats were fed with a diet containing 1, 2 or 4% di-(2-ethylhexyl)-phthalate (DEHP), a peroxisome-proliferating agent. Others were fed with normal diet as controls. Both groups were sacrificed at varying intervals from 3 days to 4 weeks. The livers were either removed and fixed in glutaraldehyde and osmium tetroxide or fixed in glutaraldehyde, incubated in a diaminobenzidine (DAB) medium, postfixed, embedded in Epon, and sectioned. Other tissues were incubated in Eaglés MEM containing either [³H]thymidine or [³H]uridine, fixed, embedded in Epon, sectioned, and radioautographed. Specimens were observed in a Hitachi H-700 electron microscope.The number of peroxisomes showing DAB reactivity increased in DEHP-fed animals as compared with normal controls In radioautograms of normal rats labelled with [³H]thymidine, no silver grains were, observed, whereas grains were observed over some nuclei, mitochondria and peroxisomes of DEHP-fed animals. In contrast, radioautograms of tissue labelled with [³H]uridine revealed a few grains in nuclei and mitochondria or endoplasmic reticulum of normal rats, although grains appeared in nuclei, mitochondria, endoplasmic reticulum and peroxisomes of DEHP-fed animals more frequently.From these results, it is concluded that [³H]thymidine and [³H]uridine were incorporated in the proliferating peroxisomes, suggesting that nucleic acid synthesis had taken place.     

The ins and outs of peroxisomes: co-ordination of membrane transport and peroxisomal metabolism

Peroxisomes perform a range of metabolic functions which require the movement of substrates, co-substrates, cofactors and metabolites across the peroxisomal membrane. In this review, we discuss the evidence for and against specific transport systems involved in peroxisomal metabolism and how these operate to co-ordinate biochemical reactions within the peroxisome with those in other compartments of the cell.     

Targeting signals in peroxisomal membrane proteins

Peroxisomal membrane proteins (PMPs) are encoded by the nuclear genome and translated on cytoplasmic ribosomes. Newly synthesized PMPs can be targeted directly from the cytoplasm to peroxisomes or travel to peroxisomes via the endoplasmic reticulum (ER). The mechanisms responsible for the targeting of these proteins to the peroxisomal membrane are still rather poorly understood. However, it is clear that the trafficking of PMPs to peroxisomes depends on the presence of cis-acting targeting signals, called mPTSs. These mPTSs show great variability both in the identity and number of requisite residues. An emerging view is that mPTSs consist of at least two functionally distinct domains: a targeting element, which directs the newly synthesized PMP from the cytoplasm to its target membrane, and a membrane-anchoring sequence, which is required for the permanent insertion of the protein into the peroxisomal membrane. In this review, we summarize our knowledge of the mPTSs currently identified.     

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)     

Analysis of the major integral membrane proteins of peroxisomes from mouse liver

Two major proteins with subunit molecular masses of 68 and 70 kDa were isolated from the integral membrane protein fraction of peroxisomes purified from mouse liver. The two proteins were shown to be distinct proteins by two criteria: first, immunoblot analysis demonstrated that antisera against the 68 kDa protein did not cross-react with the 70 kDa protein, and vice versa; and second, the partial peptide maps resulting from proteinase digestion of the proteins were different. Immunoblot analyses to test the specificities of the antisera demonstrated that only the expected molecular mass species in purified peroxisomes, and in membranes prepared from these organelles, were recognized; there was no identification of proteins from purified mitochondrial or microsomal fractions. The concentrations of both of these proteins were increased in livers of mice treated with clofibrate, a hypolipidemic drug and peroxisome proliferator, with the effect being greater for the 70 kDa component. The localization of the 68 kDa protein was shown to be completely integral to the peroxisome membrane. Although some 70 kDa protein was integral to the membrane, a significant proportion was released from the membrane by some procedures believed to detach peripheral proteins. The 70 kDa protein was also particularly susceptible to degradation during isolation - in particular, addition of EDTA to media used for isolation of peroxisomes resulted in membranes in which this protein was degraded to smaller immunoreactive fragments. These data have been discussed in relation to the significant clarification which they have provided of the status and characteristics of the major protein components of peroxisomal membranes.