Isolation and characterization of peroxisomes from the liver of normal untreated rats

The classic method of Leighton et al. [(1968) J. Cell Biol. 37, 482-513] for the isolation of peroxisomes from rat liver involves the use of Triton WR-1339 which alters the biochemical properties of this organelle and requires the specialized type Beaufay-rotor which is not easily available. We have employed Metrizamide as the gradient medium and a commercial type vertical rotor to obtain highly purified and structurally well-preserved peroxisomes from normal untreated animals. The livers were homogenized in buffered 0.25 M sucrose and a slightly modified 'light mitochondrial fraction' was prepared by differential centrifugation. This was loaded on top of a linear Metrizamide gradient (1.12-1.26 g/cm3) and subjected to an integrated force of 1.252 X 10(6) X (g X min) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at the density of 1.245 g/cm3. In the isolated fraction 95% of the protein was contributed by peroxisomes, which exhibited a strong activity for cyanide-insensitive lipid beta-oxidation. The purity of fractions was also confirmed by morphometry, which revealed that 98% of isolated particles consisted of peroxisomes. The latency for catalase was about 90% indicating a high degree of peroxisomal integrity. This corresponded to the low level of extraction of catalase in 3,3'-diaminobenzidine-stained filter preparations. The entire procedure took about five hours. Highly purified and structurally well preserved peroxisomes should be useful in further elucidation of the function of this organelle and especially in studies of peroxisomal enzymes with multiple intracellular localizations.     

Binding and functions of ADP-ribosylation factor on mammalian and yeast peroxisomes

We have analyzed in vitro the binding characteristics of members of the ADP-ribosylation factor (ARF) family of proteins to a highly purified rat liver peroxisome preparation void of Golgi membranes and studied in vivo a role these proteins play in the proliferation of yeast peroxisomes. Although both ARF1 and ARF6 were found on peroxisomes, coatomer recruitment only depended on ARF1-GTP. Recruitment of ARF1 and coatomer to peroxisomes was significantly affected both by pretreating the animals with peroxisome proliferators and by ATP and a cytosolic fraction designated the intermediate pool fraction depleted of ARF and coatomer. In the presence of ATP, the concentrations of ARF1 and coatomer on peroxisomes were reduced, whereas intermediate pool fraction led to a concentration-dependent decrease in ARF and increase in coatomer. Brefeldin A, a fungal toxin that is known to reduce ARF1 binding to Golgi membranes, did not affect ARF1 binding to peroxisomes. In Saccharomyces cerevisiae, both ScARF1 and ScARF3, the yeast orthologs of mammalian ARF1 and ARF6, were implicated in the control of peroxisome proliferation. ScARF1 regulated this process in a positive manner, and ScARF3 regulated it in a negative manner.     

Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.     

Peroxisomes move by hitchhiking on early endosomes using the novel linker protein PxdA

Eukaryotic cells use microtubule-based intracellular transport for the delivery of many subcellular cargos, including organelles. The canonical view of organelle transport is that organelles directly recruit molecular motors via cargo-specific adaptors. In contrast with this view, we show here that peroxisomes move by hitchhiking on early endosomes, an organelle that directly recruits the transport machinery. Using the filamentous fungus Aspergillus nidulans we found that hitchhiking is mediated by a novel endosome-associated linker protein, PxdA. PxdA is required for normal distribution and long-range movement of peroxisomes, but not early endosomes or nuclei. Using simultaneous time-lapse imaging, we find that early endosome-associated PxdA localizes to the leading edge of moving peroxisomes. We identify a coiled-coil region within PxdA that is necessary and sufficient for early endosome localization and peroxisome distribution and motility. These results present a new mechanism of microtubule-based organelle transport in which peroxisomes hitchhike on early endosomes and identify PxdA as the novel linker protein required for this coupling.     

Dynamin-like protein 1 is involved in peroxisomal fission

The mammalian dynamin-like protein 1 (DLP1), a member of the dynamin family of large GTPases, possesses mechanochemical properties known to constrict and tubulate membranes. In this study, we have combined two experimental approaches, induction of peroxisome proliferation by Pex11pbeta and expression of dominant-negative mutants, to test whether DLP1 plays a role in peroxisomal growth and division. We were able to localize DLP1 in spots on tubular peroxisomes in HepG2 cells. In addition, immunoblot analysis revealed the presence of DLP1 in highly purified peroxisomal fractions from rat liver and an increase of DLP1 after treatment of rats with the peroxisome proliferator bezafibrate. Expression of a dominant negative DLP1 mutant deficient in GTP hydrolysis (K38A) either alone or in combination with Pex11pbeta caused the appearance of tubular peroxisomes but had no influence on their intracellular distribution. In co-expressing cells, the formation of tubulo-reticular networks of peroxisomes was promoted, and peroxisomal division was completely inhibited. These findings were confirmed by silencing of DLP1 using siRNA. We propose a direct role for the dynamin-like protein DLP1 in peroxisomal fission and in the maintenance of peroxisomal morphology in mammalian cells.     

Cytoplasmic dynein undergoes intracellular redistribution concomitant with phosphorylation of the heavy chain in response to serum starvation and okadaic acid

Cytoplasmic dynein is a microtubule-binding protein which is considered to serve as a motor for retrograde organelle movement. In cultured fibroblasts, cytoplasmic dynein localizes primarily to lysosomes, membranous organelles whose movement and distribution in the cytoplasm have been shown to be dependent on the integrity of the microtubule cytoskeleton. We have recently identified conditions which lead to an apparent dissociation of dynein from lysosomes in vivo, indicating that alterations in membrane binding may be involved in the regulation of retrograde organelle movement (Lin, S. X. H., and C. A. Collins. 1993. J. Cell Sci. 105:579-588). Both brief serum withdrawal and low extracellular calcium levels induced this alteration, and the effect was reversed upon addition of serum or additional calcium. Here we demonstrate that the phosphorylation state of the dynein molecule is correlated with changes in its intracellular distribution in normal rat kidney fibroblasts. Dynein heavy chain phosphorylation level increased during serum starvation, and decreased back to control levels upon subsequent addition of serum. We found that okadaic acid, a phosphoprotein phosphatase inhibitor, mimicked the effects of serum starvation on both phosphorylation and the intracellular redistribution of dynein from a membrane-associated pool to one that was more soluble, with similar dose dependence for both phenomena. Cell fractionation by differential detergent extraction revealed that a higher proportion of dynein was present in a soluble pool after serum starvation than was found in comparable fractions from control cells. Our data indicate that cytoplasmic dynein is phosphorylated in vivo, and changes in phosphorylation state may be involved in a regulatory mechanism affecting the distribution of this protein among intracellular compartments.     

A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.     

Plant peroxisomes,reactive oxygen metabolism and nitric oxide

In plant cells, as in most eukaryotic organisms, peroxisomes are probably the major sites of intracellular H2O2 production, as a result of their essentially oxidative type of metabolism. Like mitochondria and chloroplasts, peroxisomes also produce superoxide radicals (O2*-) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29, and 32 kDa have been shown to generate O2*- radicals. Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the four enzymes of the ascorbate-glutathione cycle plus ascorbate and glutathione, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of plant peroxisomes. The presence of the enzyme nitric oxide synthase (NOS) and its reaction product, nitric oxide (NO*), has been recently demonstrated in plant peroxisomes. Different experimental evidence has suggested that peroxisomes have a ROS-mediated cellular function in leaf senescence and in stress situations induced by xenobiotics and heavy metals. Peroxisomes could also have a role in plant cells as a source of signal molecules like NO*, O2*- radicals, H2O2, and possibly S-nitrosoglutathione (GSNO). It seems reasonable to think that a signal molecule-producing function similar to that postulated for plant peroxisomes could also be performed by human, animal and yeast peroxisomes, where research on oxy radicals, antioxidants and nitric oxide is less advanced than in plant peroxisomes.     

The progression of peroxisomal degradation through autophagy requires peroxisomal division

Peroxisomes are highly dynamic organelles that have multiple functions in cellular metabolism. To adapt the intracellular conditions to the changing extracellular environment, peroxisomes undergo constitutive segregation and degradation. The segregation of peroxisomes is mediated by 2 dynamin-related GTPases, Dnm1 and Vps1, whereas, the degradation of peroxisomes is accomplished through pexophagy, a selective type of autophagy. During pexophagy, the size of the organelle is always a challenging factor for the efficiency of engulfment by the sequestering compartment, the phagophore, which implies a potential role for peroxisomal fission in the degradation process, similar to the situation with selective mitochondria degradation. In this study, we report that peroxisomal fission is indeed critical for the efficient elimination of the organelle. When pexophagy is induced, both Dnm1 and Vps1 are recruited to the degrading peroxisomes through interactions with Atg11 and Atg36. In addition, we found that specific peroxisomal fission, which is only needed for pexophagy, occurs at mitochondria-peroxisome contact sites.     

The progression of peroxisomal degradation through autophagy requires peroxisomal division

Peroxisomes are highly dynamic organelles that have multiple functions in cellular metabolism. To adapt the intracellular conditions to the changing extracellular environment, peroxisomes undergo constitutive segregation and degradation. The segregation of peroxisomes is mediated by 2 dynamin-related GTPases, Dnm1 and Vps1, whereas, the degradation of peroxisomes is accomplished through pexophagy, a selective type of autophagy. During pexophagy, the size of the organelle is always a challenging factor for the efficiency of engulfment by the sequestering compartment, the phagophore, which implies a potential role for peroxisomal fission in the degradation process, similar to the situation with selective mitochondria degradation. In this study, we report that peroxisomal fission is indeed critical for the efficient elimination of the organelle. When pexophagy is induced, both Dnm1 and Vps1 are recruited to the degrading peroxisomes through interactions with Atg11 and Atg36. In addition, we found that specific peroxisomal fission, which is only needed for pexophagy, occurs at mitochondria-peroxisome contact sites.     

Cytochrome P-450 2E1 in Rat Liver Peroxisomes: Downregulation by Ischemia/Reperfusion-Induced Oxidative Stress

Cytochrome P-450 containing enzymes, known to be present in the endoplasmic reticulum and mitochondria, catalyze the oxidation of various compounds. In this study we have used highly purified peroxisomes (>95%) to provide evidence by analytical cell fractionation, enzyme activity, Western blot, and immunocytochemical analysis that cytochrome P-450 2E1 (Cyp 2E1) is present in peroxisomes. Similar specific activities of aniline hydroxylase, a Cyp 2E1-dependent enzyme, in purified peroxisomes (0.72 ± 0.03 nmol/min/mg protein) and microsomes (0.58 ± 0.03 nmol/min/mg protein) supports the conclusion that peroxisomes contain significant amount of Cyp 2E1. This peroxisomal Cyp 2E1 was also induced in acetone-treated rat liver. The status of microsomal and peroxisomal Cyp 2E1 was also examined following ischemia/reperfusion-induced oxidative stress. Ischemia alone had no effect; however, reperfusion following ischemia resulted in decrease in Cyp 2E1 both in microsomes and peroxisomes. This demonstration of cytochrome P-450 2E1 in peroxisomes and its downregulation during ischemia/reperfusion describes a new role for this organelle in cytochrome P-450 related cellular metabolism and in oxidative stress induced disease conditions.     

Trypanosoma brucei: the tumor promoter thapsigargin stimulates calcium release from an intracellular compartment in slender bloodstream forms.

Maintenance of calcium homeostasis is a critical activity of eukaryotic cells. Homeostatic pathways stabilize intracellular free calcium concentrations ([Ca2+]i) at the resting level and provide the source of mobilized calcium for cellular activation. We have measured calcium release from intracellular pools within bloodstream forms of Trypanosoma brucei to better understand homeostatic pathways which operate in these organisms. Fura-2 and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to quantitate [Ca2+]i and intracellular pH (pHi), respectively. We report that the tumor promoter, thapsigargin, elevated [Ca2+]i by 50-75 nM. Mn2+ quench experiments demonstrated that the source of calcium was intracellular. No change in pHi was associated with the release of calcium from this compartment. In contrast, nigericin released approximately three-fold more calcium than thapsigargin from a pH-sensitive, intracellular pool. The nigericin-sensitive pool was nonmitochondrial. The effects of thapsigargin and nigericin on [Ca2+]i were additive, regardless of the order in which the treatment was given. We conclude that at least two pools of exchangeable calcium occur in bloodstream forms of T. brucei. One pool is sensitive to thapsigargin and apparently resides within the endoplasmic reticulum, while the nigericin-sensitive pool is nonmitochondrial and is of unknown origin.     

A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA in plant peroxisomes

Short-chain acyl-CoA oxidases are beta-oxidation enzymes that are active on short-chain acyl-CoAs and that appear to be present in higher plant peroxisomes and absent in mammalian peroxisomes. Therefore, plant peroxisomes are capable of performing complete beta-oxidation of acyl-CoA chains, whereas mammalian peroxisomes can perform beta-oxidation of only those acyl-CoA chains that are larger than octanoyl-CoA (C8). In this report, we have shown that a novel acyl-CoA oxidase can oxidize short-chain acyl-CoA in plant peroxisomes. A peroxisomal short-chain acyl-CoA oxidase from Arabidopsis was purified following the expression of the Arabidopsis cDNA in a baculovirus expression system. The purified enzyme was active on butyryl-CoA (C4), hexanoyl-CoA (C6), and octanoyl-CoA (C8). Cell fractionation and immunocytochemical analysis revealed that the short-chain acyl-CoA oxidase is localized in peroxisomes. The expression pattern of the short-chain acyl-CoA oxidase was similar to that of peroxisomal 3-ketoacyl-CoA thiolase, a marker enzyme of fatty acid beta-oxidation, during post-germinative growth. Although the molecular structure and amino acid sequence of the enzyme are similar to those of mammalian mitochondrial acyl-CoA dehydrogenase, the purified enzyme has no activity as acyl-CoA dehydrogenase. These results indicate that the short-chain acyl-CoA oxidases function in fatty acid beta-oxidation in plant peroxisomes, and that by the cooperative action of long- and short-chain acyl-CoA oxidases, plant peroxisomes are capable of performing the complete beta-oxidation of acyl-CoA.     

Intraperoxisomal redox balance in mammalian cells: oxidative stress and interorganellar cross-talk

Reactive oxygen species (ROS) are at once unsought by-products of metabolism and critical regulators of multiple intracellular signaling cascades. In nonphotosynthetic eukaryotic cells, mitochondria are well-investigated major sites of ROS generation and related signal initiation. Peroxisomes are also capable of ROS generation, but their contribution to cellular oxidation-reduction (redox) balance and signaling events are far less well understood. In this study, we use a redox-sensitive variant of enhanced green fluorescent protein (roGFP2-PTS1) to monitor the state of the peroxisomal matrix in mammalian cells. We show that intraperoxisomal redox status is strongly influenced by environmental growth conditions. Furthermore, disturbances in peroxisomal redox balance, although not necessarily correlated with the age of the organelle, may trigger its degradation. We also demonstrate that the mitochondrial redox balance is perturbed in catalase-deficient cells and upon generation of excess ROS inside peroxisomes. Peroxisomes are found to resist oxidative stress generated elsewhere in the cell but are affected when the burden originates within the organelle. These results suggest a potential broader role for the peroxisome in cellular aging and the initiation of age-related degenerative disease.     

Alcohol oxidase assembles post-translationally into the peroxisome of Candida boidinii

Candida yeasts rapidly form peroxisomes of simple function and composition when grown on methanol. Because the induction is both massive and rapid, this system may be useful for a detailed dissection of peroxisomal biogenesis. We report procedures to express peroxisomal proteins in cells and spheroplasts of Candida boidinii to stabilize peroxisomes in a lysate of spheroplasts and to obtain an enriched peroxisomal fraction. With these techniques we have been able to study the assembly of alcohol oxidase, a homo-octomeric flavoprotein, into the organelle in vivo. The primary translation product of alcohol oxidase comigrates on sodium dodecyl sulfate-polyacrylamide gels with the mature subunit. Pulse-chase experiments indicate that the newly synthesized monomer of alcohol oxidase has a half-life of about 20 min in intact cells and 13 min in spheroplasts before conversion to octomer. The monomer first appears in a high speed supernatant of a lysate of spheroplasts and then chases into a purified peroxisomal fraction before or during its octomerization. There is no detectable intermediary organelle involved in this process.     

Peroxisome division in the yeast Yarrowia lipolytica is regulated by a signal from inside the peroxisome

We describe an unusual mechanism for organelle division. In the yeast Yarrowia lipolytica, only mature peroxisomes contain the complete set of matrix proteins. These mature peroxisomes assemble from several immature peroxisomal vesicles in a multistep pathway. The stepwise import of distinct subsets of matrix proteins into different immature intermediates along the pathway causes the redistribution of a peroxisomal protein, acyl-CoA oxidase (Aox), from the matrix to the membrane. A significant redistribution of Aox occurs only in mature peroxisomes. Inside mature peroxisomes, the membrane-bound pool of Aox interacts with Pex16p, a membrane-associated protein that negatively regulates the division of early intermediates in the pathway. This interaction inhibits the negative action of Pex16p, thereby allowing mature peroxisomes to divide.     

Purification of brain peroxisomes and localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol biosynthesis. Because proper CNS development depends on de novo cholesterol biosynthesis, peroxisomes must play a critical functional role in this process. Surprisingly, no information is available on the peroxisomal isoprenoid/cholesterol biosynthesis pathway in normal brain tissue or on the compartmentalization of isoprene metabolism in the CNS. This has been due mainly to the lack of a well-defined isolation procedure for brain tissue, and also to the presence of myelin in brain tissue, which results in significant contamination of subcellular fractions. As a first step in characterizing the peroxisomal isoprenoid pathway in the CNS, we have established a purification procedure to isolate peroxisomes and other cellular organelles from the brain stem, cerebellum and spinal cord of the mouse brain. We demonstrate by use of marker enzymes and immunoblotting with antibodies against organelle specific proteins that the isolated peroxisomes are highly purified and well separated from the ER and mitochondria, and are free of myelin contamination. The isolated peroxisomal fraction was purified at least 40-fold over the original homogenate. In addition, we show by analytical subcellular fractionation and immunoelectron microscopy that HMG-CoA reductase protein and activity are localized both in the ER and peroxisomes in the CNS.     

Type II NAD(P)H dehydrogenases are targeted to mitochondria and chloroplasts or peroxisomes in Arabidopsis thaliana

We found that four type II NAD(P)H dehydrogenases (ND) in Arabidopsis are targeted to two locations in the cell; NDC1 was targeted to mitochondria and chloroplasts, while NDA1, NDA2 and NDB1 were targeted to mitochondria and peroxisomes. Targeting of NDC1 to chloroplasts as well as mitochondria was shown using in vitro and in vivo uptake assays and dual targeting of NDC1 to plastids relies on regions in the mature part of the protein. Accumulation of NDA type dehydrogenases to peroxisomes and mitochondria was confirmed using Western blot analysis on highly purified organelle fractions. Targeting of ND proteins to mitochondria and peroxisomes is achieved by two separate signals, a C-terminal signal for peroxisomes and an N-terminal signal for mitochondria.     

Peroxisomal membrane ascorbate peroxidase is sorted to a membranous network that resembles a subdomain of the endoplasmic reticulum

The peroxisomal isoform of ascorbate peroxidase (APX) is a novel membrane isoform that functions in the regeneration of NAD(+) and protection against toxic reactive oxygen species. The intracellular localization and sorting of peroxisomal APX were examined both in vivo and in vitro. Epitope-tagged peroxisomal APX, which was expressed transiently in tobacco BY-2 cells, localized to a reticular/circular network that resembled endoplasmic reticulum (ER; 3,3'-dihexyloxacarbocyanine iodide-stained membranes) and to peroxisomes. The reticular network did not colocalize with other organelle marker proteins, including three ER reticuloplasmins. However, in vitro, peroxisomal APX inserted post-translationally into the ER but not into other purified organelle membranes (including peroxisomal membranes). Insertion into the ER depended on the presence of molecular chaperones and ATP. These results suggest that regions of the ER serve as a possible intermediate in the sorting pathway of peroxisomal APX. Insight into this hypothesis was obtained from in vivo experiments with brefeldin A (BFA), a toxin that blocks vesicle-mediated protein export from ER. A transiently expressed chloramphenicol acetyltransferase-peroxisomal APX (CAT-pAPX) fusion protein accumulated only in the reticular/circular network in BFA-treated cells; after subsequent removal of BFA from these cells, the CAT-pAPX was distributed to preexisting peroxisomes. Thus, plant peroxisomal APX, a representative enzymatic peroxisomal membrane protein, is sorted to peroxisomes through an indirect pathway involving a preperoxisomal compartment with characteristics of a distinct subdomain of the ER, possibly a peroxisomal ER subdomain.     

Demonstration of Cu-Zn superoxide dismutase in rat liver peroxisomes. Biochemical and immunochemical evidence.

In this study, by using highly purified rat liver peroxisomes, we provide evidence from analytical cell fractionation, Western blot, and immunocytochemical analysis that Cu-Zn superoxide dismutase is present in animal peroxisomes. Treatment with ciprofibrate, a peroxisome proliferator, increased the peroxisomal superoxide dismutase activity by 3-fold with no effect on mitochondrial activity but a marked decrease in cytosolic superoxide dismutase activity, further supporting that besides cytosolic and mitochondrial localization, Cu-Zn superoxide dismutase is present in peroxisomes also. Demonstration of superoxide dismutase in peroxisomes suggests a new role for this organelle in pathophysiological conditions, such as ischemia-reperfusion injury.