Induction of β-oxidation enzymes and microbody proliferation in Aspergillus nidulans

Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced β-oxidation pathway showed the presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase. After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the β-oxidation enzymes, isocitrate lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies (peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase. The β-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans. Received: 8 March 1996 / Accepted: 5 August 1996     

Localization of Glyoxylate Cycle Enzymes in Glyoxysomes in Euglena*

SYNOPSIS. We demonstrated previously microbodies in Euglena gracilis grown in the dark on 2-carbon substrates. We have now established in Euglena the particulate nature of enzymes known in other organisms to be localized in microbodies (glyoxysomes and leaf peroxisomes). On a linear sucrose gradient the glyoxylate cycle enzymes band together at a nigner equilibrium density (1.20 g/cm3) than mitochondrial marker enzymes (1.17 g/cm3), establishing the existence in Euglena of glyoxysomes similar to those of higher plants. Glyoxylate (hydroxypyruvate) reductase and, under certain conditions, also glycolate dehydrogenase co-band with the glyoxylate cycle enzymes, suggesting that Euglena glyoxysomes, like those of higher plants, may contain peroxisomal-type enzymes. Catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected in Euglena.     

Microbodies in fungi: a review

Summary Microbodies are ubiquitous organelles in fungal cells, occurring in both vegetative hyphae and spores. They are bounded by a single membrane and may contain a crystalloid inclusion with subunits spaced at regular intervals. Typically, they contain catalase which reacts with the cytochemical stain 3,3-diaminobenzidine to yield an electron-opaque product, urate oxidase,l--hydroxy acid oxidase andd-amino acid oxidase. Their fragility and the necessity to disrupt the tough fungal cell wall before isolating them make them difficult to isolate. Analysis of enzymes in purified or partially purified microbodies from fungi indicates that they participate in fatty acid degradation, the glyoxylate cycle, purine metabolism, methanol oxidation, assimilation of nitrogenous compounds, amine metabolism and oxalate synthesis. In organisms where microbodies are known to contain enzymes of the glyoxylate cycle, they are known as glyoxysomes; where they are known to contain peroxidatic activity, they are known as peroxisomes. In some cases microbodies contain enzymes for only a portion of a pathway or cycle. Thus, they must be involved in metabolic cooperation with other organelles, particularly mitochondria. The number, size and shape of microbodies in cells, their buoyant density and their enzyme contents may vary with the composition of the medium; their proliferation in cells is regulated by the growth environment. The isolation from the same organism of microbodies with different buoyant densities and different enzymes suggests strongly that more than one type of microbody can be formed by fungi.     

Visualization of microbodies in Chlamydomonas reinhardtii

In Chlorophycean algal cells, these organelles are generally called microbodies because they lack the enzymes found in the peroxisomes of higher plants. Microbodies in some algae contain fewer enzymes than the peroxisomes of higher plants, and some unicellular green algae in Chlorophyceae such as Chlamydomonas reinhardtii do not possess catalase, an enzyme commonly found in peroxisomes. Thus, whether microbodies in Chlorophycean algae are similar to the peroxisomes of higher plants, and whether they use a similar transport mechanism for the peroxisomal targeting signal (PTS), remain unclear. To determine whether the PTS is present in the microbodies of Chlorophycean algae, and to visualize the microbodies in Chlamydomonas cells, we examined the sub-cellular localization of green fluorescent proteins (GFP) fused to several PTS-like sequences. We detected GFP compartments that were spherical with a diameter of 0.3-1.0?μm in transgenic Chlamydomonas. Comparative analysis of the character of GFP-compartments observed by fluorescence microscopy and that of microbodies by electron microscopy indicated that the compartments were one and the same. The result also showed that the microbodies in Chlorophycean cells have a similar transport mechanism to that of peroxisomes of higher plants.     

Investigation of cellular targeting of carotenoid pathway enzymes in Pichia pastoris

Cellular targeting of lycopene biosynthetic enzymes was investigated in Pichia pastoris X-33. Three lycopene pathway enzymes, CrtE, CrtB, and CrtI, were fused to fluorescent EGFPs with or without a peroxisomal targeting sequence (PTS1) and then expressed in P. pastoris. When P. pastoris was grown in YPD, the PTS1 fusion enzymes were found to be localized in peroxisomes, whereas the enzymes not fused with PTS1 were equally distributed throughout the entire cell. A similar targeting pattern was also observed in P. pastoris strains that were grown in peroxisome-proliferating medium, YPOT. Analysis of the fluorescent images of isolated peroxisomes showed that the PTS1 fused enzymes were dominantly present in peroxisomes whereas small amount of the enzymes not fused with PTS1 were non-specifically sent to peroxisomes. These results indicate that PTS1 specifically target lycopene pathway enzymes into peroxisomes and this targeting pathway was strong enough to overcome their inherent targeting program. In conclusion, we first showed that carotenogenic enzymes can be targeted into the specific cellular location of recombinant hosts and this targeting strategy can serve as the basis for the subsequent development of sophisticated pathway engineering in microorganisms.     

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons: Correlative Biochemical and Ultrastructural Study in Light- and Dark-grown Seedlings

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.     

Intact Cell Transformation of Saccharomyces cerevisiae by Polyethylene Glycol

The activities of isocitrate lyase and malate synthase—the key enzymes in the glyoxylate cycle—were found to be fairly high in n-alkane-, acetate-, and propionate-grown cells of Candida tropicalis compared with those in glucose-grown cells. In fact, the results of immunochemical studies showed that the increases in the enzyme levels resulted from increases in the amounts of the enzyme proteins. But the increases in these enzyme activities were not always coincident with the appearance of peroxisomes. Isocitrate lyase and malate synthase were purified from a peroxisome-containing particulate fraction of alkane-grown cells and from whole cells grown on glucose, acetate and propionate. The respective enzymes showed no significant differences in immunochemical properties, specific activities, molecular masses of active forms and subunits, on patterns of limited proteolysis with proteases, but the malate synthases of alkane- and propionate- grown cells showed higher Km values for acetyl-CoA than the enzymes of glucose- and acetate- grown cells. The results indicated that the synthesis of the key enzymes in the glyoxylate cycle did not necessarily have to be coincident with the development of peroxisomes in this yeast.     

Plant Peroxisomes: Molecular Basis of the Regulation of their Functions

β -oxidation, the glyoxylate cycle and the glycolate pathway for photorespiration. Recent molecular biological studies have revealed that most of these enzymes possess either one of two peroxisomal targeting signals (PTS) within their amino acid sequence. One of the signals, PTS1, is found at the carboxy-terminus, while the other, PTS2, is found within the amino-terminal presequence. Subsequent to the synthesis and folding of these enzymes in the cytosol, the targeting signal in the folded proteins may bind to the corresponding receptors. At present, only a receptor that recognizes PTS1 has been identified in higher plants. After the binding of the protein and the receptor, the protein complex may be recognized by docking proteins that exist in the peroxisomal membrane. The mechanisms responsible for the recognition of peroxisomal proteins are now under investigation. Genetic analyses of Arabidopsis mutants with defective peroxisomes may give us some clues to understanding the mechanisms of peroxisomal protein import. Received 18 November 1999/ Accepted in revised form 13 January 2000     

Fungal siderophore biosynthesis is partially localized in peroxisomes

Siderophores play a central role in iron metabolism and virulence of most fungi. Both Aspergillus fumigatus and Aspergillus nidulans excrete the siderophore triacetylfusarinine C (TAFC) for iron acquisition. In A. fumigatus, green fluorescence protein‐tagging revealed peroxisomal localization of the TAFC biosynthetic enzymes SidI (mevalonyl‐CoA ligase), SidH (mevalonyl‐CoA hydratase) and SidF (anhydromevalonyl‐CoA transferase), while elimination of the peroxisomal targeting signal (PTS) impaired both, peroxisomal SidH‐targeting and TAFC biosynthesis. The analysis of A. nidulans mutants deficient in peroxisomal biogenesis, ATP import or protein import revealed that cytosolic mislocalization of one or two but, interestingly, not all three enzymes impairs TAFC production during iron starvation. The PTS motifs are conserved in fungal orthologues of SidF, SidH and SidI. In agreement with the evolutionary conservation of the partial peroxisomal compartmentalization of fungal siderophore biosynthesis, the SidI orthologue of coprogen‐type siderophore‐producing Neurospora crassa was confirmed to be peroxisomal. Taken together, this study identified and characterized a novel, evolutionary conserved metabolic function of peroxisomes.     

Alkyl-dihydroxyacetonephosphate synthase

The initial steps of ether phospholipid biosynthesis take place in peroxisomes. Alkyl-dihydroxyacetonephosphate synthase, the peroxisomal enzyme that actually introduces the ether linkage, has been purified from guinea pig liver in this laboratory. With the amino acid sequences obtained from this protein, the authors were able to clone the cDNAs encoding this enzyme from both guinea pig and human liver. In both cases, the enzyme appears to be synthesized as a precursor protein with a N-terminal cleavable presequence containing a peroxisomal targeting signal (PTS) type 2. Levels of the enzyme protein were found to be strongly reduced in human fibroblasts derived from Zellweger syndrome and rhizomelic chondrodysplasia punctata patients. The molecular basis of an isolated alkyl-dihydroxyacetonephosphate synthase deficiency was resolved. A clone encoding a Caenorhabditis elegans homolog of the mammalian enzymes was characterized. In contrast to the mammalian enzymes, this C. elegans enzyme lacks a N-terminal PTS type 2 motif, but carries a C-terminal PTS type 1.     

Ultrastructure and isolation of glyoxysomes (microbodies) in zoospores of the fungusentophlyctis sp.

Summary A correlative approach, involving light and electron microscopic, cytochemical, and biochemical techniques, was used to study the structure and function of microbodies in zoospores ofEntophlyctis sp. The same population of microbodies already existing in the zoosporangium appeared to be segregated into zoospore initials during cytoplasmic cleavage. Microbodies laid at the anterior end of zoospores and were part of an organized assemblage of organelles, the microbody-lipid globule complex. In the microbody-lipid globule complex, endoplasmic reticulum occurred on the surface of the lipid globules toward the zoospore's exterior, and the microbody, subtended by mitochondria, was appressed to the opposite surface of the lipid globule. The organization of the microbody-lipid globule complex changed as the zoospore swam and encysted. As lipid globules coalesced, the microbody-lipid globule complex became disorganized. After lipid globule coalescence was completed, the microbody-lipid globule complex regained its order, and several microbodies were clustered adjacent to a single lipid globule. The microbodies persisted even in the encysted zoospore, but they were found on all sides of the lipid globule.Microbodies isolated from zoospores contained catalase as well as malate synthase and isocitrate lyase, two enzymes of the glyoxylate cycle. When zoospores encysted greater activities of these glyoxylate cycle enzymes could be detected. The presence of glyoxylate cycle enzymes and the close association between the microbody and lipid globule suggest that microbodies function as glyoxysomes in zoospores and encysted zoospores. The functional significance of the morphological organization of the microbody-lipid complex is discussed in terms of energy production and the conversion of storage lipid into structural components of the cell.     

Subcellular localization of glyoxylate cycle enzymes in Ascaris suum larvae

Evidence is presented on the particulate nature of glyoxylate cycle enzymes in metazoa with the use of 15-day old larvae of the nematode Ascaris suum. Homogenization procedures were developed to disrupt the resistant nematode cuticle. Malate synthase and isocitrate lyase, key enzymes of the glyoxylate cycle, consistently sedimented with mitochondrial enzymes in differential pellets while catalase, a major peroxisomal enzyme, was always soluble. Isopycnic sucrose gradient centrifugation of the differential pellet yielded two protein peaks: one at 1.18 g/cm3 (characteristic for mitochondria), and another at 1.23 g/cm3 (common for glyoxysomes and peroxisomes). Electron microscopy of these fractions revealed that the lighter peak consisted primarily of mitochondria, while the heavier band contained proteinaceous bodies termed "dense granules" morphologically resembling microbodies. SIgnificantly, both malate synthase and isocitrate lyase cosedimented with the mitochondrial marker enzymes in the lighter peak (1.18 g/cm3) and not with the dense granules. Further purification of mitochondria, accomplished by separating dense granules with a step gradient before isopycnic centrifugation, substantiated the evidence that microbodies (glyoxysomes) do not occur in these nematode larvae. Rough-surfaced membranes were alternatively considered as the subcellular site, but the evidence tends to favor localization of the glyoxylate bypass enzymes in the mitochondria.     

Isolation of microbodies from plant tissues

Specialized microbodies have previously been isolated and characterized from fatty seedling tissues (glyoxysomes) and leaves (leaf peroxisomes). We have now examined 11 other plant tissues, including tubers, fruits, roots, shoots, and petals, and find that all contain particulate catalase, a distinctive common enzyme component of microbodies. On linear sucrose gradients the catalase activity peaks sharply at a higher equilibrium density (1.20 to 1.25 gram per cm³ in the various tissues) than the mitochondria (1.17 to 1.20). Only small amounts of protein are recovered in the fractions containing catalase, although a definite band is visible in preparations from some tissues, e.g., potato. As in the preparations from castor bean endosperm and spinach leaves for which comparable data are provided, the distribution of glycolate oxidase and uricase follows closely that of catalase on the gradients. The preparations from potato lack glyoxylate reductase and the transaminases, typical enzymes of leaf peroxisomes, and the distinctive enzymes of glyoxysomes are missing. Nonspecialized microbodies with limited enzyme composition can thus be isolated from a variety of plant tissues.     

Distribution of peroxisomes and glycolate metabolism in relation to calcium oxalate formation in Lemna minor L

Calcium oxalate formation in Lemna minor L. occurs in structurally specialized cells called crystal idioblasts. Cytochemical and immunocytochemical protocols were employed to study the distribution of peroxisomes and the enzymes glycolate oxidase, glycine decarboxylase and ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) in relation to synthesis of oxalate used for Ca oxalate formation. These enzymes are necessary for photorespiratory glycolate synthesis and metabolism. Using catalase cytochemistry, microbodies were found to exist in crystal idioblasts but were smaller and fewer than those found in mesophyll cells. Glycolate oxidase, which can oxidize glycolate to oxalate via glyoxylate, could not be found in microbodies of crystal idioblasts at any stage of development. This enzyme increased in amount in microbodies of mesophyll cells as they matured and could even be found in dense amorphous inclusions of mature cell peroxisomes. Glycine decarboxylase and RuBisCO could also be detected in increasing amount in mesophyll cells as they matured but could not be detected in idioblasts or were just detectable. Thus, Lemna idioblasts lack the machinery for synthesis of oxalate from glycolate. Based on these results and other available information, two general models for the generation and accumulation of oxalate used for Ca oxalate formation in crystal idioblasts are proposed. The biochemical specialization of crystal idioblasts indicated by this study is also discussed with respect to differentiation of cellular structure and function.     

The control of fruiting body formation in the ascomycete Sordaria macrospora Auersw. by arginine and biotin: a two-factor analysis

Summary The distribution of glyoxylate-cycle enzymes between microbodies and mitochondria was examined in ethanol-grown Aspergillus tamarii Kita. Particulate activities of catalase and the two glyoxylate by-pass enzymes, malate synthase and isocitrate lyase, were localized in the microbodies. The microbodies had a buoyant density of about 1.23 g cm^-3 after isopycnic centrifugation in linear sucrose gradients. Particulate activities of the other two glyoxycitrate synthase, together with that of succinate dehydrogenase were restricted to the mitochondria, which had a buoyant density of about 1.20 g cm^-3. Catalase also appeared to be localized in a second particle, perhaps the microbody inclusions or the Woronin bodies, having a buoyant density of about 1.26 g cm^-3.     

The significance of peroxisome function in chronological aging of Saccharomyces cerevisiae

We studied the chronological lifespan of glucose‐grown Saccharomyces cerevisiae in relation to the function of intact peroxisomes. We analyzed four different peroxisome‐deficient (pex) phenotypes. These included Δpex3 cells that lack peroxisomal membranes and in which all peroxisomal proteins are mislocalized together with Δpex6 in which all matrix proteins are mislocalized to the cytosol, whereas membrane proteins are still correctly sorted to peroxisomal ghosts. In addition, we analyzed two mutants in which the peroxisomal location of the β‐oxidation machinery is in part disturbed. We analyzed Δpex7 cells that contain virtually normal peroxisomes, except that all matrix proteins that contain a peroxisomal targeting signal type 2 (PTS2, also including thiolase), are mislocalized to the cytosol. In Δpex5 cells, peroxisomes only contain matrix proteins with a PTS2 in conjunction with all proteins containing a peroxisomal targeting signal type 1 (PTS1, including all β‐oxidation enzymes except thiolase) are mislocalized to the cytosol. We show that intact peroxisomes are an important factor in yeast chronological aging because all pex mutants showed a reduced chronological lifespan. The strongest reduction was observed in Δpex5 cells. Our data indicate that this is related to the complete inactivation of the peroxisomal β‐oxidation pathway in these cells due to the mislocalization of thiolase. Our studies suggest that during chronological aging, peroxisomal β‐oxidation contributes to energy generation by the oxidation of fatty acids that are released by degradation of storage materials and recycled cellular components during carbon starvation conditions.     

Investigation of the glyoxysome-peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy

Microbodies in the cotyledons of cucumber seedlings perform two successive metabolic functions during early postgerminative development. During the first 4 or 5 d, glyoxylate cycle enzymes accumulate in microbodies called glyoxysomes. Beginning at about day 3, light-induced activities of enzymes involved in photorespiratory glycolate metabolism accumulate rapidly in microbodies. As the cotyledonary microbodies undergo a functional transition from glyoxysomal to peroxisomal metabolism, both sets of enzymes are present at the same time, either within two distinct populations of microbodies with different functions or within a single population of microbodies with a dual function. We have used protein A-gold immunoelectron microscopy to detect two glyoxylate cycle enzymes, isocitrate lyase (ICL) and malate synthase, and two glycolate pathway enzymes, serine:glyoxylate aminotransferase (SGAT) and hydroxypyruvate reductase, in microbodies of transition-stage (day 4) cotyledons. Double-label immunoelectron microscopy was used to demonstrate directly the co-existence of ICL and SGAT within individual microbodies, thereby discrediting the two-population hypothesis. Quantitation of protein A- gold labeling density confirmed that labeling was specific for microbodies. Quantitation of immunolabeling for ICL or SGAT in microbodies adjacent to lipid bodies, to chloroplasts, or to both organelles revealed very similar labeling densities in these three categories, suggesting that concentrations of glyoxysomal and peroxisomal enzymes in transition-stage microbodies probably cannot be predicted based on the apparent associations of microbodies with other organelles.     

CYTOCHEMICAL LOCALIZATION OF MALATE SYNTHASE IN GLYOXYSOMES

Cytochemical staining techniques for microbodies (peroxisomes) are limited at present to the enzymes catalase and α-hydroxy acid oxidase, and neither technique can distinguish glyoxysomes from other microbodies. Described here is a procedure using ferricyanide for the cytochemical demonstration by light and electron microscopy of malate synthase activity in glyoxysomes of cotyledons from fat-storing cucumber and sunflower seedlings. Malate synthase, a key enzyme of the glyoxylate cycle, catalyzes the condensation of acetyl CoA with glyoxylate to form malate and release free coenzyme A. Localization of the enzyme activity is based on the reduction by free CoA of ferricyanide to ferrocyanide, and the visualization of the latter as an insoluble, electron-opaque deposit of copper ferrocyanide (Hatchett's brown). The conditions and optimal concentrations for the cytochemical reaction mixture were determined in preliminary studies using a colorimetric assay developed to measure disappearance of ferricyanide at 420 nm. Ultrastructural observation of treated tissue reveals electron-opaque material deposited uniformly throughout the matrix portion of the glyoxysomes, with little background deposition elsewhere in the cell. The reaction product is easily visualized in plastic sections by phase microscopy without poststaining. Although the method has been applied thus far only to cotyledons of fat-storing seedlings, it is anticipated that the technique will be useful in localizing and studying glyoxylate cycle activity in a variety of tissues from both plants and animals.     

Protein targeting and import into plant peroxisomes

A main characteristic of the eucaryotic cell is the compartmentalization of different metabolic processes into membrane-enclosed organelles. Each organelle contains a characteristic set of proteins to accomplish specific metabolic functions that are often essential for the cell's viability. The most recently discovered class of organelles includes the microbodies that encompass a group of organelles which have some morphological properties in common. Microbodies are ubiquitous in eucaryotic cells and can be subdivided into different types of organelles according to their metabolic functions (e.g. peroxisomes and glyoxysomes). The size and number of microbodies per cell is often related to the developmental stage and/or the organism in which they occur. This implies that microbody proliferation is inductible in nature. This review summarizes the progress made in recent years in understanding how proteins are targeted to and imported into microbodies. Major breakthroughs were the identification of the two main peroxisomal protein targeting signals (PTS1 and PTS2), protein receptors for the signals and the isolation of yeast mutants defective in the biogenesis of microbodies. Especially the availability of these mutants has opened new ways to identify proteins involved in microbody protein import in plants as well as animals.     

A correlative ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds

Summary Sunflower, cucumber, and tomato cotyledons, which contain microbodies in both the early lipid-degrading and the later photosynthetic stages of post-germinative growth, were processed for electron microscopy according to conventional procedures and examined 1, 4 and 7 days after germination. Homogenates of sunflower cotyledons were assayed for enzymes characteristic of glyoxysomes and leaf peroxisomes (both of which are defined morphologically as microbodies) at stages corresponding to the fixations for electron microscopy. The particulate nature of these enzymes was demonstrated by differential and equilibrium density centrifugation, making it possible to relate them to the microbodies seen in situ.One day after germination, the microbodies are present as small organelles among large numbers of protein and lipid storage bodies; the cell homogenate contains catalase but no detectable isocitrate lyase (characteristic of glyoxysomes) or glycolic acid oxidase (characteristic of leaf peroxisomes). 4 days after germination, numerous microbodies (glyoxysomes) are in extensive and frequent contact with lipid bodies. The microbodies often have cytoplasmic invaginations. At this stage the cells are rapidly converting lipids to carbohydrates, and the homogenate has high isocitrate lyase activity. 7 days after germination, microbodies (peroxisomes) are appressed to chloroplasts and frequently squeezed between them in the green photosynthetic cells. The homogenate at this stage has substantial glycolic acid oxidase activity but a reduced level of isocitrate lyase. It is yet to be determined whether the peroxisomes present at day 7 are derived from preexisting glyoxysomes or arise as a separate population of organelles.