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.     

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.     

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats.

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.     

Biogenesis of peroxisomes: immunocytochemical investigation of peroxisomal membrane proteins in proliferating rat liver peroxisomes and in catalase-negative membrane loops

Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554-564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment.     

Membranes of rat liver peroxisomes

Summary Membranes of liver peroxisomes from rats fed with clofibrate were purified in a discontinuous gradient using a zonal rotor. The preparation consists of round or oval vesicles mostly devoid of nucleoids with a diameter ranging from 70–700 nm; open sheets are found very infrequently. Mitochondrial profiles as well as vesicles containing cytochemically demonstrable glucose 6-phosphatase are scarce; accordingly, glucose 6-phosphatase is nearly undetectable biochemically. Monoamine oxidase is absent in peroxisomal membranes. Cytochrome b₅ is found in a concentration of 0.3 nmoles/mg protein, an order of magnitude comparable to the content of endoplasmic reticulum membranes. Reduction of this cytochrome with palmitoyl-CoA is possible only after recombination of the membranes with the soluble peroxisomal matrix fraction.     

Functional and structural membrane topology of rat liver microsomal glutathione transferase

The membrane topology of rat liver microsomal glutathione transferase was investigated by comparing the tryptic cleavage products from intact and permeabilized microsomes. It was shown that lysine-4 of microsomal glutathione transferase is accessible at the luminal surface of the endoplasmic reticulum, whereas lysine-41 faces the cytosol. These positions are separated by a hydrophobic stretch of 25 amino acids (positions 11–35) which comprises the likely membrane-spanning region. Reaction of cysteine-49 of the microsomal glutathione transferase with the charged sulfhydryl reagent DTNB (2,2′-dithiobis(5-nitrobenzoic acid))) in intact microsomes further supports the cytosolic localization of this portion of the polypeptide chain. The role of two other potential membrane-spanning/associated segments in the C-terminal half of the polypeptide chain was examined by investigating the association of the protein to the membrane after trypsin cleavage at lysine-41. Activity measurements and Western blot analysis after washing with high concentrations of salt, as well as after phase separation in Triton X-114, indicate that this portion of the protein also binds to the membrane. It is also shown that cleavage of the purified protein at Lys-41 and subsequent separation of the fragments obtained yields a functional C-terminal polypeptide with the expected length for the product encompassing positions 42–154. The location of the active site of microsomal glutathione transferase was investigated using radiolabelled glutathione together with a second substrate. Since isolated rat liver microsomes do not take up glutathione or release the glutathione conjugate into the lumen, it can be concluded that the active site of rat liver microsomal glutathione transferase faces the cytosolic side of the endoplasmic reticulum.     

Freeze-fracture analysis of isolated liver mitochondria in different metabolic states

Isolated liver mitochondria were crosslinked with glutaraldehyde during different respiratory states and then analyzed either by freeze-fracturing, or in thin sections prepared according to a low denaturation embedding technique. In freeze-fractured material, large intracristal spaces were found in 86% of the mitochondria freshly isolated in a sucrose medium. These results were in good agreement with mitochondria analyzed in thin sections, in which case 88% of the mitochondria contained intracristal spaces. In freeze-fractured mitochondria, only 24% of the mitochondria crosslinked after 3 min in respiratory state 4 contained intracristal spaces, while 36% of the mitochondria contained intracristal spaces during state 3. These values compared favorably with those obtained in embedded material in which case 21 and 41% of the mitochondria contained intracristal spaces during states 3 and 4, respectively. The agreement between the observations made with the two methods demonstrated that the disappearance of intracristal spaces is independent of the embedding technique, and is not a special effect caused by dehydration in ethylene glycol.     

Glycerolipid synthetic capacity of rat liver peroxisomes

Investigations on rat liver peroxisomal glycerolipid synthetic capability were performed. Highly purified peroxisomal preparations contained dihydroxyacetone-phosphate acyltransferase, acyldihydroxyacetone-phosphate reductase, and fatty acid-CoA ligase activities. Glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, diacylglycerol cholinephosphotransferase, diacylglycerol ethanolaminephosphotransferase and ethanol acyltransferase activities were low in activity or not detected. These results suggest that the peroxisomes are specialized to contribute to the synthesis of ether-linked glycerolipids. If peroxisomes contribute towards the synthesis of non-ether-linked glycerolipids (i.e., ester-linked) then translocation of acyl glycerophosphatide (acyl dihydroxyacetone phosphatide) from peroxisomes to endoplasmic reticulum would be expected to occur.     

Degradation of cholesterol to propionic acid by rat liver peroxisomes

Under standard assay conditions peroxisomes were found to contain less than 5% of the liver's cholesterol degradation activity. The remainder of the activity was localized in the mitochondria. When CaCl₂ was added to the standard assay mixture, peroxisomal cholesterol degradation activity increased to 34%. These results suggest that peroxisomes are capable of cholesterol catabolism, with the assay conditions used invitro determining the relative organelle contribution.     

Biosynthesis of dolichol by rat liver peroxisomes

The ability of peroxisomes and microsomes to synthesize dolichol from [3H]mevalonate, [3H]isopentenyl-P2 or [3H]farnesyl-P2 in vitro was investigated. It was found that isoprenoid biosynthesis also occurs in peroxisomes and that this process demonstrates properties differing from those of isoprenoid biosynthesis by microsomes. The pH optimum in peroxisomes was 8.0 and, in contrast to microsomes, the peroxisomal biosynthesis was largely insensitive to detergents. After treatment with proteolytic enzymes, microsomes lost their capacity to incorporate [3H]mevalonate into dolichol, whereas proteolysis of intact peroxisomes did not influence their corresponding rate of incorporation. The soluble content of peroxisomes was separated from the membranes and found to demonstrate half of the biosynthetic capacity of the intact organelle. Fasting and cholestyramine treatment decreased only the microsomal incorporation of [3H]mevalonate into dolichol, while treatment with clofibrate, di-2-ethylhexyl phthalate or phenobarbital increased microsomal, but decreased peroxisomal labeling. After injection of [3H]mevalonate into the portal vein of rats, high initial labeling of dolichol was recovered both in isolated microsomes and peroxisomes, whereas when [3H]glycerol was administered, peroxisomal phospholipids became labeled later than the corresponding microsomal constituents. These results support the conclusion that dolichol is synthesized both in peroxisomes and the endoplasmic reticulum, but that the biosynthetic processes at these two locations have different properties.     

Subcellular localization and membrane topology of sphingosine-1-phosphate lyase in rat liver

Sphingosine-1-phosphate lyase is responsible for the ultimate step in sphingolipid breakdown, converting phosphorylated long chain bases into ethanolamine phosphate and a fatty aldehyde. Using tritiated dihydrosphingosine-1-phosphate, prepared enzymatically from [4,5-3H]dihydrosphingosylphosphocholine, we have reinvestigated the subcellular distribution of this enzyme in rat liver. Upon cell fractionation by differential centrifugation, the enzyme showed a microsomal distribution. Further separation of the microsomal fraction by sucrose gradient centrifugation confirmed an association with the endoplasmic reticulum. By means of constrained nonlinear regression, no evidence for a significant association with mitochondrial membranes, as reported previously (Stoffel, W., LeKim, D., and Sticht, G. (1969) Hoppe Seyler's Z. Physiol. Chem. 350, 1233-1241), nor with other cell compartments was found. The lyase activity, which appeared to be sensitive to different detergents, but not to Triton X-100, was not latent. It could be solubilized with Triton X-100, but not by high ionic strength, indicating that it is an integral membrane protein whose catalytic site is most probably exposed to the cytosol. Treatment of intact microsomal vesicles with trypsin or thermolysin inactivated the lyase activity, confirming that its catalytic site(s) or other domains essential for activity face the cytosol.     

Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: comparison with endoplasmic reticulum and mitochondrial membranes

Membranes were isolated from highly purified peroxisomes, mitochondria, and rough and smooth microsomes of rat liver by the one-step Na2CO3 procedure described in the accompanying paper (1982, J. Cell Biol. 93:97-102). The polypeptide compositions of these membranes were determined by SDS PAGE and found to be greatly dissimilar. The peroxisomal membrane contains 12% of the peroxisomal protein and consists of three major polypeptides (21,700, 67,700 and 69,700 daltons) as well as some minor polypeptides. The major peroxisomal membrane proteins as well as most of the minor ones are absent from the endoplasmic reticulum (ER). Conversely, most ER proteins are absent from peroxisomes. By electron microscopy, purified peroxisomal membranes are approximately 6.8 nm thick and have a typical trilaminar appearance. The phospholipid/protein ratio of peroxisomal membranes is approximately 200 nmol/mg; the principal phospholipids are phosphatidyl choline and phosphatidyl ethanolamine as in ER and mitochondrial membranes. In contrast to the mitochondria, peroxisomal membranes contain no cardiolipin. All the membranes investigated contain a polypeptide band with a molecular mass of approximately 15,000 daltons. Whether this represents an exceptional common membrane protein or a coincidence is unknown. The implications of these results for the biogenesis of peroxisomes are discussed.     

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.     

Insights into the membrane proteome of rat liver peroxisomes: microsomal glutathione-S-transferase is shared by both subcellular compartments

Peroxisomes are ubiquitous "multipurpose" organelles of eukaryotic cells. Their matrix enzymes catalyze mainly catabolic and anabolic reactions of lipid metabolism, thus contributing to the regulation of lipid homeostasis. Since most metabolites must be actively transported across the peroxisomal membrane and since individual proteins and protein complexes play functional roles in such transport processes, we analyzed the peroxisomal membrane proteome. Benzyldimethyl-n-hexadecylammoniumchloride (16-BAC)/SDS-2-D-PAGE and mass spectrometry were used to characterize the proteomes of highly purified "light" and "heavy" peroxisomes of rat liver obtained by density gradient centrifugation. In both populations, the major integral membrane proteins could be detected in high concentrations, verifying 16-BAC/SDS-2-D-PAGE as a suitable tool for the preparation of membrane proteomes destined for mass spectrometric analysis. Both reliable and reproducible detection of a distinct set of microsomal (ER) membrane proteins, including microsomal glutathione-S-transferase (mGST), in light and heavy peroxisomal fractions was also possible. Compared with the abundance of most microsomal membrane proteins, we found mGST to be specifically enriched in peroxisomal membrane fractions. Furthermore, C terminus epitope-tagged mGST versions were localized at least in part to peroxisomes in different mammalian cell lines. Taken together, these data suggest that the peroxisomal GST is not a mere ER-contaminant, but a bona fide protein comprising the membrane proteome of both intracellular compartments. In addition, we could detect several mitochondrial proteins in light peroxisome fractions. This finding may likely indicate a physical association of light peroxisomes with mitochondria, since the organelles could be partly separated by mechanical stress. Whether this association is of functional importance awaits further investigation.     

Two-dimensional gel electrophoretic resolution of the polypeptides of rat liver mitochondria and the outer membrane

The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.     

Chemical modification of rat liver microsomal cytochrome P-450: study of enzymic properties and membrane topology

Isolated rat liver cytochrome P-450IIB1 was alkylated and acetylated at primary amino groups, and the position of the modified amino acids in the protein was identified. Alkylation of up to nine amino groups did not disturb the interaction of reconstituted P-450 and NADPH-cytochrome-P-450 reductase in a way that hydroxylation of benzphetamine was altered, whereas deethylation of 7-ethoxycoumarin was gradually reduced in parallel with impaired 7-ethoxycoumarin binding. Acetylation of four lysine residues completely inhibited binding and metabolism of 7-ethoxycoumarin but not of benzphetamine. These results suggest the presence of different substrate binding sites on P-450. Exhaustive proteolysis of modified P-450 in proteoliposomes liberated all but the N-terminal modified peptide and 85 to 90% of the cytochrome's mass from intact proteoliposomes. These findings further support our previously proposed model of P-450 topology (Vergères, G., Winterhalter, K.H. and Richter, C. (1989) Biochemistry 28, 3650-3655), in which P-450 is anchored to the membrane with the N-terminal peptide only, the N-terminal methionine facing the lumenal interior.     

Phosphoinositide synthesis and degradation in isolated rat liver peroxisomes

Analyzing peroxisomal phosphoinositide (PId(#)) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P(2) and PtdIns(3,5)P(2). PtdIns3P was hardly detected in vitro, however, was observed in vivo after [(32)P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of phosphatase inhibitors enhanced the amount of PtdIns4P. The results obtained provide evidence that isolated rat liver peroxisomes synthesize PIds and suggest the association of PId 4-kinase and PId 5-kinase and PId 4-phosphatase activities with the peroxisomal membrane.     

Two different targeting signals direct human peroxisomal membrane protein 22 to peroxisomes

The 22-kDa peroxisomal membrane protein (PMP22) is a major component of peroxisomal membranes in mammals. Although its precise role in peroxisome function is poorly understood, it seems to be involved in pore forming activity and may contribute to the unspecific permeability of the organelle membrane. PMP22 is synthesized on free cytosolic ribosomes and then directed to the peroxisome membrane by specific targeting information. Previous studies in rats revealed that PMP22 contains one distinct peroxisomal membrane targeting signal in the amino-terminal cytoplasmic tail. We cloned and characterized the targeting signal of human PMP22 and compared it with the already described characteristics of the corresponding rat protein. Amino acid sequence alignment of rat and human protein revealed 77% identity including a high conservation of several protein motifs. We expressed various deletion constructs of PMP22 in fusion with the green fluorescent protein in COS-7 cells and determined their intracellular localization. In contrast to previous studies on rat PMP22 and most other peroxisomal membrane proteins, we showed that human as well as rat PMP22 contains two distinct and nonoverlapping peroxisomal membrane targeting signals, one in the amino-terminal and the other in the carboxyl-terminal protein region. They consist of two transmembrane domains and adjacent protein loops with almost identical basic clusters. Both of these peroxisomal targeting regions interact with PEX19, a factor required for peroxisome membrane synthesis. In addition, we observed that fusing the green fluorescent protein immediately adjacent to the targeting region completely abolishes targeting function and mislocalizes PMP22 to the cytosol.     

Manganese superoxide dismutase in rat liver peroxisomes: Biochemical and immunochemical evidence

By using highly purified peroxisomes from rat liver, we have shown that peroxisomes contain manganese superoxide dismutase (MnSOD) activity and a 23 kDa protein immunoreactive with antibodies against purified mitochondrial MnSOD. Immunocytochemical studies have also revealed immunoreaction (immunogold) with MnSOD antibodies in mitochondria and peroxisomes. Studies of the intraperoxisomal localization of MnSOD have shown that in peroxisomes MnSOD is a component of the peroxisomal limiting membranes and dense core. Furthermore, the MnSOD level in peroxisomes was modulated by oxidative stress conditions such as ischemia-reperfusion or the treatment with ciprofibrate, a peroxisomal proliferator. These findings suggest that MnSOD in peroxisomes may play an important role in the dismutation of superoxide generated on the peroxisomal membrane for keeping the delicate balance of the redox state.