Turnover Of Peroxisomes Research Articles

Segregation of pathways leading to pexophagy.

Peroxisomes are organelles with key roles in metabolism including long-chain fatty acid production. Their metabolic functions overlap and interconnect with those of mitochondria, with which they share an overlapping but distinct proteome. Both organelles are degraded by selective autophagy processes termed pexophagy and mitophagy. Although mitophagy has received intense attention, the pathways linked to pexophagy and associated tools are less well developed. We have identified the neddylation inhibitor MLN4924 as a potent activator of pexophagy and show that this is mediated by the HIF1α-dependent up-regulation of BNIP3L/NIX, a known adaptor for mitophagy. We show that this pathway is distinct from pexophagy induced by the USP30 deubiquitylase inhibitor CMPD-39, for which we identify the adaptor NBR1 as a central player. Our work suggests a level of complexity to the regulation of peroxisome turnover that includes the capacity to coordinate with mitophagy, via NIX, which acts as a rheostat for both processes.

TP53 loss-of-function causes vulnerability to autophagy inhibition in aggressive prostate cancer.

TP53 loss-of-function is commonly found in aggressive prostate cancer. However, a highly-efficient therapy for this tumor subtype is still lacking. In this study, we investigated the relationship between TP53 mutation status and autophagy in prostate cancer and assessed the efficacy of autophagy inhibitors on TP53-deficient tumors. We first evaluated the expression patterns of p53 and autophagy-related proteins, namely LC3B, ULK1 and BECLIN1, as well as their relationship in treatment-naïve and castration-resistant prostate cancer specimens through immunohistochemistry. Subsequently, we generated a Trp53-deleted genetically-engineered mouse model, established prostate tumor organoid lines from the mice and assessed the efficacy of autophagy inhibitors in overcoming Enzalutamide resistance in the tumor organoid model. We also investigated the impact of TP53 re-expression in modulating responses to autophagy inhibitors using LNCaP cell line, which harbored a TP53 missense mutation. Lastly, we attempted to identify potential autophagy-related genes that were crucial for TP53-deficient tumor maintenance. TP53 loss-of-function was associated with increased levels of autophagy-related proteins in aggressive prostate cancers and Trp53-deleted genetically-engineered mouse-derived tumors. Moreover, the generated androgen receptor-independent tumor organoids were highly vulnerable to autophagy inhibition. Upon TP53 re-expression, not only did the surviving LNCaP cells demonstrate resistance, but they also showed growth advantage in response to autophagy inhibition. Lastly, PEX14, an important peroxisomal regulator was differentially upregulated in aggressive tumors with TP53 loss-of-function mutations, thus implying the importance of peroxisome turnover in this tumor subtype. Our results support the potential use of autophagy inhibitors in prostate cancers that contain TP53 loss-of-function mutations.

Pexophagy is critical for fungal development, stress response, and virulence in Alternaria alternata.

Alternaria alternata can resist high levels of reactive oxygen species (ROS). The protective roles of autophagy or autophagy‐mediated degradation of peroxisomes (termed pexophagy) against oxidative stress remain unclear. The present study, using transmission electron microscopy and fluorescence microscopy coupled with a GFP‐AaAtg8 proteolysis assay and an mCherry tagging assay with peroxisomal targeting tripeptides, demonstrated that hydrogen peroxide (H2O2) and nitrogen depletion induced autophagy and pexophagy. Experimental evidence showed that H2O2 triggered autophagy and the translocation of peroxisomes into the vacuoles. Mutational inactivation of the AaAtg8 gene in A. alternata led to autophagy impairment, resulting in the accumulation of peroxisomes, increased ROS sensitivity, and decreased virulence. Compared to the wild type, ΔAaAtg8 failed to detoxify ROS effectively, leading to ROS accumulation. Deleting AaAtg8 down‐regulated the expression of genes encoding an NADPH oxidase and a Yap1 transcription factor, both involved in ROS resistance. Deleting AaAtg8 affected the development of conidia and appressorium‐like structures. Deleting AaAtg8 also compromised the integrity of the cell wall. Reintroduction of a functional copy of AaAtg8 in the mutant completely restored all defective phenotypes. Although ΔAaAtg8 produced wild‐type toxin levels in axenic culture, the mutant induced a lower level of H2O2 and smaller necrotic lesions on citrus leaves. In addition to H2O2, nitrogen starvation triggered peroxisome turnover. We concluded that ΔAaAtg8 failed to degrade peroxisomes effectively, leading to the accumulation of peroxisomes and the reduction of the stress response. Autophagy‐mediated peroxisome turnover could increase cell adaptability and survival under oxidative stress and starvation conditions.

Plant peroxisome proteostasis-establishing, renovating, and dismantling the peroxisomal proteome.

Plant peroxisomes host critical metabolic reactions and insulate the rest of the cell from reactive byproducts. The specialization of peroxisomal reactions is rooted in how the organelle modulates its proteome to be suitable for the tissue, environment, and developmental stage of the organism. The story of plant peroxisomal proteostasis begins with transcriptional regulation of peroxisomal protein genes and the synthesis, trafficking, import, and folding of peroxisomal proteins. The saga continues with assembly and disaggregation by chaperones and degradation via proteases or the proteasome. The story concludes with organelle recycling via autophagy. Some of these processes as well as the proteins that facilitate them are peroxisome-specific, while others are shared among organelles. Our understanding of translational regulation of plant peroxisomal protein transcripts and proteins necessary for pexophagy remain based in findings from other models. Recent strides to elucidate transcriptional control, membrane dynamics, protein trafficking, and conditions that induce peroxisome turnover have expanded our knowledge of plant peroxisomal proteostasis. Here we review our current understanding of the processes and proteins necessary for plant peroxisome proteostasis—the emergence, maintenance, and clearance of the peroxisomal proteome.

Benchmarking a highly selective USP30 inhibitor for enhancement of mitophagy and pexophagy.

The deubiquitylase USP30 is an actionable target considered for treatment of conditions associated with defects in the PINK1-PRKN pathway leading to mitophagy. We provide a detailed cell biological characterization of a benzosulphonamide molecule, compound 39, that has previously been reported to inhibit USP30 in an in vitro enzymatic assay. The current compound offers increased selectivity over previously described inhibitors. It enhances mitophagy and generates a signature response for USP30 inhibition after mitochondrial depolarization. This includes enhancement of TOMM20 and SYNJ2BP ubiquitylation and phosphoubiquitin accumulation, alongside increased mitophagy. In dopaminergic neurons, generated from Parkinson disease patients carrying loss of function PRKN mutations, compound 39 could significantly restore mitophagy to a level approaching control values. USP30 is located on both mitochondria and peroxisomes and has also been linked to the PINK1-independent pexophagy pathway. Using a fluorescence reporter of pexophagy expressed in U2OS cells, we observe increased pexophagy upon application of compound 39 that recapitulates the previously described effect for USP30 depletion. This provides the first pharmacological intervention with a synthetic molecule to enhance peroxisome turnover.

Dimethyloxaloylglycine induces pexophagy in a HIF-2α dependent manner involving autophagy receptor p62

Peroxisomes are metabolically active oxygen demanding organelles with a high abundance of oxidases making it vulnerable to low oxygen levels such as hypoxic conditions. However, the exact mechanism of peroxisome degradation in hypoxic condition remains elusive. In order to study the mechanism of peroxisome degradation in hypoxic condition, we use Dimethyloxaloylglycine (DMOG), a cell-permeable prolyl-4-hydroxylase inhibitor, which mimics hypoxic condition by stabilizing hypoxia-inducible factors. Here we report that DMOG degraded peroxisomes by selectively activating pexophagy in a HIF-2α dependent manner involving autophagy receptor p62. Furthermore, DMOG not only increased peroxisome turnover by pexophagy but also reduced HIF-2α dependent peroxisome proliferation at the transcriptional level. Taken together, our data suggest that hypoxic condition is a negative regulator for peroxisome abundance through increasing pexophagy and decreasing peroxisome proliferation in HIF-2α dependent manner.

Peroxisome turnover and diurnal modulation of antioxidant activity in retinal pigment epithelia utilizes microtubule-associated protein 1 light chain 3B (LC3B).

The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the retinoid cycle, nutrient supply, ion exchange, and waste removal. Each day the RPE removes the oldest ~10% of photoreceptor outer segment (OS) disk membranes through phagocytic uptake, which peaks following light onset. Impaired degradation of phagocytosed OS material by the RPE can lead to toxic accumulation of lipids, oxidative tissue damage, inflammation, and cell death. OSs are rich in very long chain fatty acids, which are preferentially catabolized in peroxisomes. Despite the importance of lipid degradation in RPE function, the regulation of peroxisome number and activity relative to diurnal OS ingestion is relatively unexplored. Using immunohistochemistry, immunoblot analysis, and catalase activity assays, we investigated peroxisome abundance and activity at 6 AM, 7 AM (light onset), 8 AM, and 3 PM, in wild-type (WT) mice and mice lacking microtubule-associated protein 1 light chain 3B (Lc3b), which have impaired phagosome degradation. We found that catalase activity, but not the amount of catalase protein, is 50% higher in the morning compared with 3 PM, in RPE of WT, but not Lc3b-/-, mice. Surprisingly, we found that peroxisome abundance was stable during the day in RPE of WT mice; however, numbers were elevated overall in Lc3b-/- mice, implicating LC3B in autophagic organelle turnover in RPE. Our data suggest that RPE peroxisome function is regulated in coordination with phagocytosis, possibly through direct enzyme regulation, and may serve to prepare RPE peroxisomes for daily surges in ingested lipid-rich OS.

Peroxisomes and Oxidative Stress: Their Implications in the Modulation of Cellular Immunity During Mycobacterial Infection.

Host redox dependent physiological responses play crucial roles in the determination of mycobacterial infection process. Mtb explores oxygen rich lung microenvironments to initiate infection process, however, later on the bacilli adapt to oxygen depleted conditions and become non-replicative and unresponsive toward anti-TB drugs to enter in the latency stage. Mtb is equipped with various sensory mechanisms and a battery of pro- and anti-oxidant enzymes to protect themselves from the host oxidative stress mechanisms. After host cell invasion, mycobacteria induces the expression of NADPH oxidase 2 (NOX2) to generate superoxide radicals (), which are then converted to more toxic hydrogen peroxide (H2O2) by superoxide dismutase (SOD) and subsequently reduced to water by catalase. However, the metabolic cascades and their key regulators associated with cellular redox homeostasis are poorly understood. Phagocytosed mycobacteria en route through different subcellular organelles, where the local environment generated during infection determines the outcome of disease. For a long time, mitochondria were considered as the key player in the redox regulation, however, accumulating evidences report vital role for peroxisomes in the maintenance of cellular redox equilibrium in eukaryotic cells. Deletion of peroxisome-associated peroxin genes impaired detoxification of reactive oxygen species and peroxisome turnover post-infection, thereby leading to altered synthesis of transcription factors, various cell-signaling cascades in favor of the bacilli. This review focuses on how mycobacteria would utilize host peroxisomes to alter redox balance and metabolic regulatory mechanisms to support infection process. Here, we discuss implications of peroxisome biogenesis in the modulation of host responses against mycobacterial infection.

Analysis of Peroxisome Biogenesis in Pollen by Confocal Microscopy and Transmission Electron Microscopy.

Peroxisome is an essential single-membrane bound organelle in most eukaryotic cells and functions in diverse cellular processes. De novo formation, division, and turnover of peroxisomes contribute to its biogenesis, morphology, and population regulation. In plants, peroxisome plays multiple roles, including metabolism, development, and stress response. Defective peroxisome biogenesis and development retard plant growth, adaption, and reproduction. Through tracing the subcellular localization of fluorescent reporter tagged matrix protein of peroxisome, fluorescence microscopy is a reliable and fast way to detect peroxisome biogenesis. Further fine-structural observation of peroxisome by TEM enables researchers to observe the detailed ultrastructure of its morphology and spatial contact with other organelles. Pollen grain is a specialized structure where two small sperm cells are enclosed in the cytoplasm of a large vegetative cell. Two features make pollen grain a good system to study peroxisome biogenesis: indispensable requirement of peroxisome for germination on the stigma and homogeneity. Here, we describe the methods of studying peroxisome biogenesis in Arabidopsis pollen grains by fluorescent live-imaging with confocal laser scanning microscopy (CLSM) and by DAB-staining based transmission electron microscopy (TEM).

Hypoxia signaling pathways: modulators of oxygen-related organelles.

Oxygen (O2) is an essential substrate in cellular metabolism, bioenergetics, and signaling and as such linked to the survival and normal function of all metazoans. Low O2 tension (hypoxia) is a fundamental feature of physiological processes as well as pathophysiological conditions such as cancer and ischemic diseases. Central to the molecular mechanisms underlying O2 homeostasis are the hypoxia-inducible factors-1 and -2 alpha (HIF-1α and EPAS1/HIF-2α) that function as master regulators of the adaptive response to hypoxia. HIF-induced genes promote characteristic tumor behaviors, including angiogenesis and metabolic reprogramming. The aim of this review is to critically explore current knowledge of how HIF-α signaling regulates the abundance and function of major O2-consuming organelles. Abundant evidence suggests key roles for HIF-1α in the regulation of mitochondrial homeostasis. An essential adaptation to sustained hypoxia is repression of mitochondrial respiration and induction of glycolysis. HIF-1α activates several genes that trigger mitophagy and represses regulators of mitochondrial biogenesis. Several lines of evidence point to a strong relationship between hypoxia, the accumulation of misfolded proteins in the endoplasmic reticulum, and activation of the unfolded protein response. Surprisingly, although peroxisomes depend highly on molecular O2 for their function, there has been no evidence linking HIF signaling to peroxisomes. We discuss our recent findings that establish HIF-2α as a negative regulator of peroxisome abundance and suggest a mechanism by which cells attune peroxisomal function with O2 availability. HIF-2α activation augments peroxisome turnover by pexophagy and thereby changes lipid composition reminiscent of peroxisomal disorders. We discuss potential mechanisms by which HIF-2α might trigger pexophagy and place special emphasis on the potential pathological implications of HIF-2α-mediated pexophagy for human health.

Hif-2α Promotes Degradation of Mammalian Peroxisomes by Selective Autophagy

Peroxisomes play a central role in lipid metabolism, and their function depends on molecular oxygen. Low oxygen tension or von Hippel-Lindau (Vhl) tumor suppressor loss is known to stabilize hypoxia-inducible factors alpha (Hif-1α and Hif-2α) to mediate adaptive responses, but it remains unknown if peroxisome homeostasis and metabolism are interconnected with Hif-α signaling. By studying liver-specific Vhl, Vhl/Hif1α, and Vhl/Hif2α knockout mice, we demonstrate a regulatory function of Hif-2α signaling on peroxisomes. Hif-2α activation augments peroxisome turnover by selective autophagy (pexophagy) and thereby changes lipid composition reminiscent of peroxisomal disorders. The autophagy receptor Nbr1 localizes to peroxisomes and is likewise degraded by Hif-2α-mediated pexophagy. Furthermore, we demonstrate that peroxisome abundance is reduced in VHL-deficient human clear cell renal cell carcinomas with high HIF-2α levels. These results establish Hif-2α as a negative regulator of peroxisome abundance and metabolism and suggest a mechanism by which cells attune peroxisomal function with oxygen availability.

Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation

In contrast to the enormous advances made regarding mechanisms of conventional protein secretion, mechanistic insights into the unconventional secretion of proteins are lacking. Acyl coenzyme A (CoA)-binding protein (ACBP; AcbA in Dictyostelium discoideum), an unconventionally secreted protein, is dependent on Golgi reassembly and stacking protein (GRASP) for its secretion. We discovered, surprisingly, that the secretion, processing, and function of an AcbA-derived peptide, SDF-2, are conserved between the yeast Pichia pastoris and D. discoideum. We show that in yeast, the secretion of SDF-2-like activity is GRASP dependent, triggered by nitrogen starvation, and requires autophagy proteins as well as medium-chain fatty acyl CoA generated by peroxisomes. Additionally, a phospholipase D implicated in soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor-mediated vesicle fusion at the plasma membrane is necessary, but neither peroxisome turnover nor fusion between autophagosomes and the vacuole is essential. Moreover, yeast Acb1 and several proteins required for its secretion are necessary for sporulation in P. pastoris. Our findings implicate currently unknown, evolutionarily conserved pathways in unconventional secretion.

Peroxisome Dynamics in Cultured Mammalian Cells

Despite the identification and characterization of various proteins that are essential for peroxisome biogenesis, the origin and the turnover of peroxisomes are still unresolved critical issues. In this study, we used the HaloTag technology as a new approach to examine peroxisome dynamics in cultured mammalian cells. This technology is based on the formation of a covalent bond between the HaloTag protein--a mutated bacterial dehalogenase which is fused to the protein of interest--and a synthetic haloalkane ligand that contains a fluorophore or affinity tag. By using cell-permeable ligands of distinct fluorescence, it is possible to image distinct pools of newly synthesized proteins, generated from a single genetic HaloTag-containing construct, at different wavelengths. Here, we show that peroxisomes display an age-related heterogeneity with respect to their capacity to incorporate newly synthesized proteins. We also demonstrate that these organelles do not exchange their protein content. In addition, we present evidence that the matrix protein content of pre-existing peroxisomes is not evenly distributed over new organelles. Finally, we show that peroxisomes in cultured mammalian cells, under basal growth conditions, have a half-life of approximately 2 days and are mainly degraded by an autophagy-related mechanism. The implications of these findings are discussed.

PpAtg30 Tags Peroxisomes for Turnover by Selective Autophagy

Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction with peroxins, and during pexophagy it colocalizes transiently at the preautophagosomal structure (PAS) and interacts with the autophagy machinery. PpAtg30 is required for formation of pexophagy intermediates, such as the micropexophagy apparatus (MIPA) and the pexophagosome (Ppg). During pexophagy, PpAtg30 undergoes multiple phosphorylations, at least one of which is required for pexophagy. PpAtg30 overexpression stimulates pexophagy even under peroxisome-induction conditions, impairing peroxisome biogenesis. Therefore, PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy.

Excess Peroxisomes Are Degraded by Autophagic Machinery in Mammals

Peroxisomes are degraded by autophagic machinery termed "pexophagy" in yeast; however, whether this is essential for peroxisome degradation in mammals remains unknown. Here we have shown that Atg7, an essential gene for autophagy, plays a pivotal role in the degradation of excess peroxisomes in mammals. Following induction of peroxisomes by a 2-week treatment with phthalate esters in control and Atg7-deficient livers, peroxisomal degradation was monitored within 1 week after discontinuation of phthalate esters. Although most of the excess peroxisomes in the control liver were selectively degraded within 1 week, this rapid removal was exclusively impaired in the mutant liver. Furthermore, morphological analysis revealed that surplus peroxisomes, but not mutant hepatocytes, were surrounded by autophagosomes in the control. Our results indicated that the autophagic machinery is essential for the selective clearance of excess peroxisomes in mammals. This is the first direct evidence for the contribution of autophagic machinery in peroxisomal degradation in mammals.

Macropexophagy in Hansenula polymorpha: facts and views

The hallmark of eukaryotic cells is compartmentalization of distinct cellular functions into specific organelles. This necessitates the cells to run energetically costly mechanisms to precisely control maintenance and function of these compartments. One of these continuously controls organelle activity and abundance, a process termed homeostasis. Yeast peroxisomes are favorable model systems for studies of organelle homeostasis because both the proliferation and degradation of these organelles can be readily manipulated. Here, we highlight recent achievements in regulation of peroxisome turnover in yeast, in particular Hansenula polymorpha, with a focus on directions of future research.

Degradation of normal and proliferated peroxisomes in rat hepatocytes: regulation of peroxisomes quantity in cells.

Degradation and turnover of peroxisomes is reviewed. First, we describe the historical aspects of peroxisome degradation research and the two major concepts for breakdown of peroxisomes, i.e., autophagy and autolysis. Next, the comprehensive knowledge on autophagy of peroxisomes in mammalian and yeast cells is reviewed. It has been shown that proliferated peroxisomes are degraded by selective autophagy, and studies using yeast cells have been especially helpful in shedding light on the molecular mechanisms of this process. The degradation of extraperoxisomal urate oxidase crystalloid is noted. Overexpressed wild-type urate oxidase in cultured cells has been shown to be degraded through an unknown proteolytic pathway distinct from the lysosomal system including autophagy or the ubiquitin-proteasome system. Finally, peroxisome autolysis mediated by 15-lipoxygenase (15-LOX) is described. 15-LOX is integrated into the peroxisome membrane causing focal membrane disruptions. The content of the peroxisomes is then exposed to cytosol proteases and seems to be digested quickly. In conclusion, the number of peroxisomes appears to be regulated by two selective pathways, autophagy, including macro- and microautophagy, and 15-LOX-mediated autolysis.

Sensitive and real-time determination of H2O2 release from intact peroxisomes.

Peroxisomes are essential and ubiquitous cell organelles having a key role in mammalian lipid and oxygen metabolism. The presence of flavine oxidases makes them an important intracellular source of H(2)O(2): an obligate product of peroxisomal redox reactions and a key reactive oxygen species. Peroxisomes proliferate in response to external signals triggered by peroxisome-proliferator-activated receptor signalling pathways. Peroxisome-derived oxidative stress as a consequence of this proliferation is increasingly recognized to participate in pathologies ranging from carcinogenesis in rodents to alcoholic and non-alcoholic steatosis hepatitis in humans. To date, no sensitive approach exists to record H(2)O(2) turnover of peroxisomes in real time. Here, we introduce a sensitive chemiluminescence method that allows the monitoring of H(2)O(2) generation and degradation in real time in suspensions of intact peroxisomes. Importantly, removal, as well as release of, H(2)O(2) can be assessed at nanomolar, non-toxic concentrations in the same sample. Owing to the kinetic properties of catalase and oxidases, H(2)O(2) forms fast steady-state concentrations in the presence of various peroxisomal substrates. Substrate screening suggests that urate, glycolate and activated fatty acids are the most important sources for H(2)O(2) in rodents. Kinetic studies imply further that peroxisomes contribute significantly to the beta-oxidation of medium-chain fatty acids, in addition to their essential role in the breakdown of long and very long ones. These observations establish a direct quantitative release of H(2)O(2) from intact peroxisomes. The experimental approach offers new possibilities for functionally studying H(2)O(2) metabolism, substrate transport and turnover in peroxisomes of eukaryotic cells.

Sensitive and real-time determination of H2O2 release from intact peroxisomes

Peroxisomes are essential and ubiquitous cell organelles having a key role in mammalian lipid and oxygen metabolism. The presence of flavine oxidases makes them an important intracellular source of H2O2: an obligate product of peroxisomal redox reactions and a key reactive oxygen species. Peroxisomes proliferate in response to external signals triggered by peroxisome-proliferator-activated receptor signalling pathways. Peroxisome-derived oxidative stress as a consequence of this proliferation is increasingly recognized to participate in pathologies ranging from carcinogenesis in rodents to alcoholic and non-alcoholic steatosis hepatitis in humans. To date, no sensitive approach exists to record H2O2 turnover of peroxisomes in real time. Here, we introduce a sensitive chemiluminescence method that allows the monitoring of H2O2 generation and degradation in real time in suspensions of intact peroxisomes. Importantly, removal, as well as release of, H2O2 can be assessed at nanomolar, non-toxic concentrations in the same sample. Owing to the kinetic properties of catalase and oxidases, H2O2 forms fast steady-state concentrations in the presence of various peroxisomal substrates. Substrate screening suggests that urate, glycolate and activated fatty acids are the most important sources for H2O2 in rodents. Kinetic studies imply further that peroxisomes contribute significantly to the β-oxidation of medium-chain fatty acids, in addition to their essential role in the breakdown of long and very long ones. These observations establish a direct quantitative release of H2O2 from intact peroxisomes. The experimental approach offers new possibilities for functionally studying H2O2 metabolism, substrate transport and turnover in peroxisomes of eukaryotic cells.

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)