Isolation and characterization of peroxisomes from the renal cortex of beef, sheep, and cat

The isolation and characterization of highly purified and structurally well-preserved peroxisomes from the renal cortex of different mammalian species (beef, sheep, and cat) is reported. Renal cortex tissue was homogenized and a peroxisome-enriched light mitochondrial fraction was prepared by differential centrifugation. This was subfractionated by density-dependent banding on a linear gradient of metrizamide (1.12-1.26 g/cm3) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at a mean density of 1.225 cm3. Ultrastructural morphometric examination revealed that peroxisomes made up 97 to 98% of the isolated fractions. By biochemical analysis the contamination with marker enzymes of mitochondria and lysosomes was extremely low. The specific activity of catalase was enriched, depending on the species, between 28- and 38-fold over the homogenate. Peroxisome preparations from all three species exhibited a high but varying level of activity for cyanide-insensitive lipid beta-oxidation. In beef and sheep preparations a small amount of esterase activity cosediments with peroxisomes. These peroxisomes show distinct structural membrane associations with smooth elements of ER. Urate oxidase, a marker enzyme for rat liver peroxisomes, is found only in peroxisomes prepared from beef kidney cortex, with sheep and cat preparations being negative. This correlated with the occurrence of polytubular inclusions in the beef kidney peroxisomes. The large size and the angular shape of isolated peroxisomes as well as the presence of paracrystalline matrical inclusions imply that the majority of peroxisomes are derived from the epithelial cells of the proximal tubule of the kidney cortex. The significant differences found in the characteristics of the renal peroxisomes in three different species investigated, demonstrate the remarkable adaptability and plasticity of this organelle.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

Immunoelectron microscopic localization of D-amino acid oxidase in rat kidney and liver

Summary The intracellular localization ofd-amino acid oxidase in rat kidney and liver has been investigated using the indirect immunogold postembedding technique. Different fixation and embedding conditions for optimal preservation of antigenicity and fine structure have been tested. Immunolabelling was possible only in tissues embedded in polar resins (glycol methacrylate and Lowicryl K4M). In kidney the enzyme was demonstrable only in the peroxisomes of the proximal tubule, where it was associated with the peroxisome core. The enzyme was present in all the peroxisomes of the proximal tubule and appeared to be codistributed with catalase. Control experiments and quantitative analysis confirmed the specificity of thed-amino acid oxidase immunolocalization. All the other cells in kidney failed to demonstrate any labelling. In liver, the immunolabelling was present in the matrix of the hepatocyte peroxisomes, whereas no traces of the enzyme were found in the nucleoid. The intensity of the immunolabelling in liver peroxisomes was lower than in kidney. No specific labelling was observed in cells other than hepatocytes.     

Cytochemical analysis of organelle degradation in phagosomes and apoptotic cells of the mucoid epithelium of mice.

The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2-6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes.     

Cytochemical analysis of organelle degradation in phagosomes and apoptotic cells of the mucoid epithelium of mice

Summary The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2–6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes.     

Cytochemical localization by ferricyanide reduction of α-hydroxy acid oxidase activity in peroxisomes of rat kidney

Summary Conditions are described for the use of ferricyanide as an electron acceptor for the cytochemical demonstration by light and electron microscopy of mammalian L--hydroxy acid oxidase activity in peroxisomes of rat kidney. Enzyme activity survives brief fixation in cold formaldehyde or in Karnovsky's fixative. Cytochemical localization of -hydroxy acid oxidase activity in cryostat sections, or in finely chopped tissue blocks, is based on a simulaneous coupling reaction, in which ferrocyanide (produced by the enzymatic reduction of ferricyanide) is captured by copper to yield an insoluble, amorphous, electron-opaque deposit of cupric ferrocyanide (Hatchett's Brown). Under cytochemical conditions, the enzyme is most active in the presence of D,L--hydroxy butyric acid. The staining reaction requires the presence of substrate, and is abolished by heat treatment of sections. The use of rubeanic acid (dithiooxamide) is recommended for the visualization of the copper-containing reaction product by light microscopy. The cytochemical localization obtained is specific for peroxisomes located in cells of the proximal tubule of the rat nephron. By light microscopy, renal peroxisomes can be distinguished from lysosomes and mitochondria on the basis of their size, shape, number, and intracellular distribution. At an ultrastructural level, amorphous, electronopaque cupric ferrocyanide reaction product is precisely localized to the nucleoid and peripheral portion of the matrix of the peroxisome in lightly stained areas, and throughout the organelle, where staining is more intense. Staining results with the ferricyanide method for L--hydroxy acid oxidase, reported herein, are compared with those obtainable with the tetrazolium technic developed by Alien and Beard for the same enzyme, and with the 3,3-diamino-benzidine (DAB) method for catalase.This study was supported by grants MT-1273 and MT-1341 from the Medical Research Council of Canada.     

Ultrastructural changes of renal tubules in the riboflavin deficient mouse

Ultrastructural changes of the tubular epithelium in the mouse kidney produced by dietary riboflavin deficiency were studied by electron microscopy and cytochemistry. In riboflavin deficient mouse kidney, the ultrastructural changes are localized to the pars recta of the proximal tubule. They comprise so called vacuolar degeneration on light microscopy, which consists of the formation of giant mitochondria and vacuoles. During the development of riboflavin deficiency, mitochondria decrease in number and enlarge in size through fusion. Sometimes they are larger than nuclei in size. The vacuoles observed in tubular epithelia are divided into two different groups according to their morphological characteristics and origins. One is derived from proliferated peroxisomes, and another from increased cytoplasmic bodies termed cytosomes and cytosegresomes. These increased vacuoles occupy almost all of cytoplasm. Cytochemical studies also reveal that these vacuoles are peroxisomes and lysosomes. These changes are reversible on supplementation with riboflavin.     

The effect of sex hormones on the proximal tubules in the rat kidney

Summary The three segments (S₁, S₂, S₃) of the proximal tubule of the rat kidney were investigated, with special reference to lysosomes, after castration, estradiol application, and at the end of pregnancy. Especially in S₁ and S₂ castration induces an increase of cellular autophagy. The nuclei become smaller; endoplasmic reticulum (ER), ribosomes, and Golgi apparatus are reduced; catabolism predominates. In S₁ more giant lysosomes occur; the total number of lysosomes increases whereas acid phosphatase activity decreases at the same time. Sex differences which exist in untreated animals disappear. Substitution with estradiol causes an activation of the proximal tubule cells: Heterophagy predominates, and cellular autophagy is reduced. Nuclear size is unchanged; ER, ribosomes and Golgi apparatus show a clear increase. Giant lysosomes are absent in S₁. On the whole lysosomes are larger, but less numerous than after castration. Acid phosphatase is highly active. All changes are most evident in S₃. At the end of pregnancy the proximal tubule cells are stressed considerably: Pinocytotic activity increases, and large numbers of cell organelles and many lipid vacuoles can be observed. The basal lamina in S₁ and S₂ becomes thicker. Lysosomes enlarge and increase in number in all segments; giant lysosomes are absent in S₁; acid phosphatase activity is extremely high. The results indicate that sex hormones directly influence the regulation of the proximal tubule cell; moreover, they are indirectly important for the functioning of the kidney via changes in the whole organism.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)Dedicated to Prof. Dr. O. Bucher, Head of the Institute of Histology and Embryology of the University of Lausanne/Switzerland, on the occasion of his 65th birthday     

Immunocytochemical localization of D-amino acid oxidase in the central clear matrix of rat kidney peroxisomes

Light and electron microscopic localizations of D-amino acid oxidase (DAO) in rat kidney was investigated using immunoenzyme and protein A-gold techniques. The enzyme was purified from rat kidney homogenate and its antibody was raised in rabbits. By Ouchterlony double-diffusion analysis and immunoblot analysis with anti-(rat kidney DAO) immunoglobulin, the antibody was confirmed to be monospecific. The tissue sections (200 micron thick) of fixed rat kidney were embedded in Epon or Lowicryl K4M. Semi-thin sections were stained for DAO by the immunoenzyme technique after removal of epoxy resin for LM, and ultra-thin sections of Lowicryl-embedded material were labeled for DAO by the protein A-gold technique for EM. By LM, fine cytoplasmic granules of proximal tubule were stained exclusively. Among three segments of proximal tubules, and S2 and S3 segments were heavily stained but the S1 segment only weakly so. By EM, gold particles indicating the antigenic sites for DAO were exclusively confined to peroxisomes. Within peroxisomes, the gold particles were localized in the central clear matrix but not in the peripheral tubular substructures. The results indicate that D-amino acid oxidase in rat kidney is present exclusively in peroxisomes in the proximal tubule and that within peroxisomes it is found only in central clear matrix and not in the peripheral tubular substructures.     

Development of peroxisomes in amphibians. III. Study on liver, kidney, and intestine during thyroxine-induced metamorphosis

This investigation was undertaken to study the ontogeny of hepatic, renal, and intestinal peroxisomes and/or microperoxisomes during thyroxine-induced anuran metamorphosis. Catalase activity was localized cytochemically after incubation in DAB medium, and studied biochemically by a spectrophotometric method. Our morphological and biochemical investigations suggest the formation of a new population of peroxisomes during the hormonal treatment. This is obvious especially for microperoxisomes of the intestinal epithelium since the larval tissue is completely replaced by a new layer during thyroxine-induced metamorphosis. For the peroxisomes of hepatocytes and kidney proximal tubule cells, our assumption is based on the following observations: 1) The number of peroxisomes increases in liver and kidney during thyroxine treatment; 2) this proliferation is accompanied by an enlargement of renal peroxisomes; and 3) 16 days after the beginning of the hormonal treatment, 5.4- and 2.4-fold increases are found for the specific activities of hepatic and renal catalase, respectively. A temporal coordination exists between the structure and the metabolism of peroxisomes and mitochondria during thyroxine-induced metamorphosis.     

Quantitative isolation of liver mitochondria by zonal centrifugation

A method was developed using zonal centrifugation to recover liver mitochondria quantiatively and free of other cellular components from a sample of whole homogenate. The fractions containing mitochondria were identified by the distribution of cytochrome oxidase and these fractions contained over 90% of the total cytochrome oxidase recovered. The mitochondrial fractions were found to be only slightly contaminated by 5′-nucleotidase (plasma membranes), acid phosphatase (lysosomes), glucose-6-phosphatase (microsomes), and catalase (peroxisomes). There was no detectable contamination by nuclear DNA (nuclei). This method was used to quantitate total liver mitochondrial protein. The development of this procedure provides a means for following total changes in mitochondrial components during mitochondrial biogenesis.     

The different effects of the peroxisome proliferators clofibric acid and bis(2-ethylhexyl)phthalate on the activities of peroxisomal oxidases in rat liver

Treatment with peroxisome proliferators induces increased numbers and alterations in the shape of peroxisomes in liver. It ultimately leads to hepatocellular carcinomas induced by the persistent production of high amounts of H2O2 as a result of a dramatical increase in acyl-CoA oxidase activity. The effects of peroxisome proliferators on other peroxisomal oxidase activities are less well documented. In the present study, the distribution patterns of the activity of SdD-amino acid oxidase, SlD-alpha-hydroxy acid oxidase, polyamine oxidase, urate oxidase and catalase activities were investigated in unfixed cryostat sections of liver, kidney and duodenum of rats treated with either clofibrate or bis(2-ethylhexyl)phthalate. The activities of xanthine oxidoreductase, which produces urate, a potent anti-oxidant, and xanthine oxidase, which produces oxygen radicals, were studied as well. The liver was the only organ that was affected by treatment. The number of peroxisomes increased considerably. SdD-Amino acid oxidase and polyamine oxidase activities were completely abolished by the treatment, whereas SlD-alpha-hydroxy acid oxidase activity decreased and urate oxidase activity increased periportally and decreased pericentrally. Total catalase activity increased because of the larger numbers of peroxisomes, but it decreased per individual peroxisome. Xanthine oxidoreductase activity decreased, whereas the percentage of xanthine oxidase remained constant. We conclude that oxidases in rat liver are affected differentially, indicating that the expression of activity of each oxidase is regulated individually. © 1998 Chapman & Hall     

Histochemical,autoradiographic and electron microscopic investigations of the renal proximal tubule of male and female rats after castration

Summary After castration of 90-day-old male and female rats, changes appear in the renal proximal tubule. A distinction can be made between early changes (up to 10th postoperative day) and later changes (20th–30th postoperative day). Between the 3rd and 5th day after castration the kidney of the females shows an increase in free estrogen receptors (biochemical studies) which are localized in the pars contorta of the proximal tubule (autoradiographic studies), while the male kidney shows a marked increase in urinary protein excretion up to the 10th day after castration. Proximal tubule changes detectable histochemically and electron microscopically do not appear until day 20 or 30 after castration. The results of castration are similar in segments S₁ and S₂. By days 20 and 30 after castration there is a decrease in the activity of lysosomal enzymes (acid phosphatase, acid -galactosidase). Electron microscopy shows a conspicuous decrease in the number of giant lysosomes (mainly in females) and apical vacuoles (mainly in males). A marked increase in the number of lysosomes is found in the S₃ segment; females always have more lysosomes than males. The number of peroxisomes is also greatly increased; they appear circular in the females but can assume bizarre shapes in the males. Lipid droplets appear in the basal region of the tubule cell of segments S₂ and S₃ in the males. The sex differences are preserved in all segments even after castration and become even more pronounced in the S₃ segment.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)Dedicated to Prof. Dr. W. Graumann, Department of Anatomy, University of Tübingen, on the occasion of his 65th birthday     

DISTRIBUTION OF PEROXISOMES (MICROBODIES) IN THE NEPHRON OF THE RAT : A Cytochemical Study

The distribution of peroxisomes (microbodies) in the rat nephron was studied cytochemically, using glutaraldehyde- or formaldehyde-fixed tissue, by means of α-hydroxy acid oxidase activity in light microscopy or oxidation of 3,3'-diaminobenzidine (DAB) at pH 9 in both light and electron microscopy.The two cytochemical methods show peroxisomes to be nearly sperical particles found only in cells of the proximal convoluted tubule. Lysosomes were identified in the same or parallel sections, with β-glycerophosphate or 5'-cytidylic acid as substrate. They are found in all cells of the nephron. These cytochemical methods visualize the two organelles for light microscopy; they also permit unequivocal differentiation of all kidney peroxisomes from lysosomes in electron micrographs. Peroxisomes are larger and more reactive in the cells of the pars descendens (P₃ segment) of the proximal convolution, located in the outer medulla and medullary rays, than in the cells of the pars convoluta (P₁ and P₂ segments), situated in the cortex. In contrast, lysosomes are much smaller in the P₃ segment and larger and more reactive in the P₁ and P₂ segments. In all cells of the proximal convolution, peroxisomes tend to be concentrated nearer the base of the cells than do lysosomes. Mitochondria in P₃ cells also show low levels of DAB oxidation at pH 6, in contrast to those in P₁ and P₂ cells. The possibility is discussed that P₃ cells possess an extramitochondrial means of oxidation in which peroxisome oxidases play an important role.     

SUBCELLULAR DISTRIBUTION OF d-AMINO ACID OXIDASE AND CATALASE IN RAT BRAIN

—Rat brain d -amino acid oxidase was found to be confined to the hindbrain, distributed more or less equally between cerebellum and medulla. Histochemical staining showed the enzyme to be localized largely in the molecular layer of the cerebellum. After fractionation by means of several distinct density gradient centrifugation procedures exploiting differences in sedimentation coefficient or in density or in both, the enzyme was found to be entirely or almost entirely associated with cytoplasmic particles with a median diameter of the order of 0·2 μm, and a median equilibrium density in aqueous sucrose of 1·18. Comparison with the behavior of cytochrome oxidase and of N-acetyl-β-glucosaminidase indicates that these particles are not mitochondria and are unlikely to be lysosomes. They also differ significantly from the bulk of the catalase-containing particles, which on an average appear to be somewhat smaller. The possibility that they might contain some catalase activity, and thereby qualify as ‘peroxisomes’, can however not be excluded. In any case, they differ profoundly from the peroxisomes of liver or kidney.     

Biogenesis and metabolic significance of microbodies in urate-utilizing yeasts

Growth of Candida famata and Trichosporon cutaneum on uric acid as the sole source of carbon and nitrogen was associated with the development of a number of microbodies in the cells. Cytochemical staining experiments showed that the organelles contained urate oxidase, a key enzyme of uric acid metabolism, and catalase. Transfer of cells, precultured on glucose or glycerol, into uric acid-containing media indicated that these microbodies originated from the organelles, originally present in the inoculum cells, by growth and division. In urate-grown C. famata the microbodies were frequently observed in large clusters; in both organisms they existed in close association with mitochondria and strands of ER. The organelles lacked crystalline inclusions. In freeze-fractured cells their surrounding membranes showed smooth fracture faces.Exposure of urate-grown cells to glucose-excess conditions led to a rapid inactivation of urate oxidase activity but catalase was only slightly inactivated. Glucose-induced enzyme inactivation was not associated with the degradation of the microbodies present in the cells. Similarly, repression of urate oxidase synthesis by ammonium ions also did not lead to the degradation of peroxisomes.     

Structure, composition, physical properties, and turnover of proliferated peroxisomes. A study of the trophic effects of Su-13437 on rat liver

Peroxisome proliferation has been induced with 2-methyl-2-(p-[1,2,3,4-tetrahydro-1-naphthyl]-phenoxy)-propionic acid (Su-13437). DNA, protein, cytochrome oxidase, glucose-6-phosphatase, and acid phosphatase concentrations remain almost constant. Peroxisomal enzyme activities change to approximately 165%, 50%, 30%, and 0% of the controls for catalase, urate oxidase, L-alpha-hydroxy acid oxidase, and D-amino acid oxidase, respectively. For catalase the change results from a decrease in particle-bound activity and a fivefold increase in soluble activity. The average diameter of peroxisome sections is 0.58 +/- 0.15 mum in controls and 0.73 +/- 0.25 mum after treatment. Therefore, the measured peroxisomal enzymes are highly diluted in proliferated particles. After tissue fractionation, approximately one-half of the normal peroxisomes and all proliferated peroxisomes show matric extraction with ghost formation, but no change in size. In homogenates submitted to mechanical stress, proliferated peroxisomes do not reveal increased fragility; unexpectedly, Su-13437 stabilizes lysosomes. Our results suggest that matrix extraction and increased soluble enzyme activities result from transmembrane passage of peroxisomal proteins. The changes in concentration of peroxisomal oxidases and soluble catalase after Su-13437 allow the calculation of their half-lives. These are the same as those found for total catalase, in normal and treated rats, after allyl isopropyl acetamide: about 1.3 days, a result compatible with peroxisome degradation by autophagy. A sequential increase in liver RNA concentration, [14C]leucine incorporation into DOC-soluble proteins and into immunoprecipitable catalase, and an increase in liver size and peroxisomal volume per gram liver, characterize the trophic effect of the drug used. In males, Su-13437 is more active than CPIB, another peroxisome proliferation-inducing drug; in females, only Su-13437 is active.     

Subcellular Distribution of Enzymes in Ochromonas malhamensis*

SYNOPSIS. The activity and distribution of 7 enzymes in Ochromonas malhamensis were studied. Subcellular organelles were separated by centrifugation at 648,000 g min to precipitate the larger particles; the resulting supernatant was centrifuged at 5,560,000 g min to separate the microsomal fraction from the supernatant. Sixty-four percent of the cytochrome oxidase (1.9.3.1 ferrocytochrome c:oxygen oxidoreductase, 81% of the catalase (1.11.1.6 hydrogen-peroxide: hydrogen-peroxide oxidoreductase) and 70% of the urate oxidase (1.7.3.3 urate:oxygen oxidoreductase) activity was associated with the larger particles, altho only 20% of the total protein was found in this fraction. Three acid hydrolases, cathepsin (3.4.4.9 cathepsin C, acid phosphatase (3.1.3.2 orthophosphoric monoesterphosphohydrolase) and acid ribonuclease (2.7.7.17 ribonucleate nucleotido-2′-transferase) were found mostly in the supernate (50-60%, yet their latency and their similar subcellular distribution indicated the presence of lysosomes. After 2.5 hr centrifugation in a sucrose density gradient (ρ= 1.08–1.25, the acid hydrolases showed a broad distribution which differed greatly from cytochrome oxidase associated with mitochondria. Catalase, which could not be separated from cytochrome oxidase by centrifuging on this gradient, had a different distribution after centrifugation on a kinetic gradient. Urate oxidase had a similar distribution to catalase and both these enzymes were latent, indicating the presence of peroxisomes.     

Cytochemical and immunocytochemical study on the peroxisomes of rat liver after administration of a hypolipidemic drug, MLM-160

After administration of a hypolipidemic drug, MLM-160, to male rats, liver peroxisomes were studied by biochemical, cytochemical, and immunocytochemical methods. The activities of D-amino acid oxidase, glycolate oxidase, and urate oxidase increased 2 to 3-fold by the treatment. The increase of the oxidases was confirmed by immunoblotting analysis. By light microscopy, immunoreaction for catalase was present in the cytoplasmic granules of hepatocytes. The stained granules formed some clusters and overlapped each other after MLM-160 treatment. However, immunostaining for D-amino acid oxidase and urate oxidase was present in discrete fine granules which did not overlap each other. By electron microscopy, many peroxisomes showed ring-like extensions and cavitation of the matrix, often giving the appearance of a peroxisome-within-a-peroxisome. In many cases, these unusual peroxisomes seemed to be interconnected with each other. Within the peroxisomes, the catalase was localized in the matrix. Urate oxidase was associated with the crystalloid cores. D-amino acid oxidase was localized focally in a small part of the matrix where the catalase was mostly negative. In conclusion, the administration of MLM-160 to male rats induces some peroxisomal oxidases, accompanying the appearance of unusual peroxisomes. The precise localization of peroxisomal enzymes suggested that there are subcompartments within the liver peroxisomes as shown in rat kidney peroxisomes.     

Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin

Mutations in the mitochondrial kinase PINK1 and the cytosolic E3 ligase Parkin can cause Parkinson's disease. Damaged mitochondria accumulate PINK1 on the outer membrane where, dependent on kinase activity, it recruits and activates Parkin to induce mitophagy, potentially maintaining organelle fidelity. How PINK1 recruits Parkin is unknown. We show that endogenous PINK1 forms a 700 kDa complex with the translocase of the outer membrane (TOM) selectively on depolarized mitochondria whereas PINK1 ectopically targeted to the outer membrane retains association with TOM on polarized mitochondria. Inducibly targeting PINK1 to peroxisomes or lysosomes, which lack a TOM complex, recruits Parkin and activates ubiquitin ligase activity on the respective organelles. Once there, Parkin induces organelle selective autophagy of peroxisomes but not lysosomes. We propose that the association of PINK1 with the TOM complex allows rapid reimport of PINK1 to rescue repolarized mitochondria from mitophagy, and discount mitochondrial-specific factors for Parkin translocation and activation.