Microbody-like Organelles Research Articles

3-Hydroxy-3-methyl-glutaryl-CoA Reductase inTrypanosoma(Schizotrypanum)cruzi:Subcellular Localization and Kinetic Properties

The subcellular localization of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, which catalyzes the first committed step of the mevalonate pathway, was investigated inTrypanosoma cruziepimastigotes using well-established cell fractionation procedures. It was found that ca. 80% of the activity of the enzyme was associated with the glycosomes, microbody-like organelles unique to kinetoplastid protozoa which contain most of the enzymes of the glycolytic pathway, while the rest of the activity was found in the soluble (cytoplasmatic) fraction, with almost no activity associated with microsomes. The glycosome-associated enzyme is not membrane-bound as it was recovered quantitatively in the aqueous phase of the biphasic system formed by Triton X-114 at 30°C. Studies with digitonin-permeabilized intact epimastigotes demonstrated the presence of two pools of soluble HMG-CoA reductase in these cells, associated to the cytoplasmic and glycosomal compartments. Steady-state kinetic studies of the glycosome-associated enzyme indicated classical Michaelis–Menten behavior withKm,app(HMG-CoA) 28 ± 3 μM,Km,app(NADPH) 37 ± 4 μM, andVm,app3.9 ± 0.2 nmol/min mg protein; the transition-state analog lovastatin behaved as a competitive inhibitor with respect to HMG-CoA withKis23 nM and a noncompetitive inhibitor toward NADPH withKii29 nM. The results are in complete agreement with recent gene cloning and expression studies which showed thatT. cruziHMG-CoA reductase lacks the NH2-terminal membrane-spanning sequence. This is the first demonstration of a soluble eukaryotic HMG-CoA reductase and also the first report on the presence of an enzyme of the isoprenoid biosynthesis pathway in glycosomes.

The fine structure and selected histochemistry of ungerminated basidiospores ofAgrocybe acericola

The basidiospore wall of Agrocybe acericola is composed of two distinct layers that are continuous around the spores. At the germ pore, the outer wall is very thin and the inner wall becomes thicker. The plasma membrane is appressed to the inner wall and lacks distinct invaginations. The protoplasm is densely packed with ribosomes. Spores contain very little lipid distributed at each end. Mitochondria are well defined and distributed throughout the cytoplasm. Spores are binucleate, with the two nuclei lying on a line nearly perpendicular to the long axis of the cell. Various sizes of single membrane-bound vacuoles are widely distributed in the cytoplasm. These vacuoles were shown to contain acid phosphatase, indicating lysosomal activity. Microbody-like organelles are observed, which are probably glyoxysomes, since assays of malate synthase, a marker enzyme of the glyoxylate cycle, are positive. Keywords: Agrocybe, spore wall ultrastructure, basidiospore ultrastructure, glyoxylate cycle, malate synthase, acid phosphatase.

Structure, function, and biogenesis of glycosomes in Kinetoplastida

Glycosomes are intracellular, microbody-like organelles found in all members of the protist order Kinetoplastida examined. Nine enzymes involved in glucose and glycerol metabolism are associated with these organelles. These enzymes are involved in pathways which, in other organisms, are usually located in the cytosol. This paper reviews our current knowledge about the glycosome and its constituent enzymes, with special reference to the organelle of Trypanosoma brucei.

Triose-phosphate isomerase of Leishmania mexicana mexicana Cloning and characterization of the gene, overexpression in Escherichia coli and analysis of the protein

The gene of triose-phosphate isomerase in Leishmania mexicana has been cloned and characterized. The gene encodes a polypeptide of 251 amino acids, with a calculated molecular mass of 27,561 Da and a net charge of +2. Only one gene could be detected, although the enzyme is present in two different compartments of the cell, in microbody-like organelles called glycosomes and in the cytosol. The primary structure of the enzyme has many features in common with that of triose-phosphate isomerase in the related organism Trypanosoma brucei. Their sequences are 68% identical. The residues constituting the subunit interface are highly conserved between the enzyme of L. mexicana and T. brucei, but are mostly different from those in the enzyme of other organisms. One major substitution was detected in the interface region of the L. mexicana protein: a glutamate was found at position 66, instead of glutamine in all other available 20 sequences. The glutamine is thought to be important for the stability of the dimeric enzyme. L. mexicana triose-phosphate isomerase has been overexpressed in Escherichia coli. Growth conditions were established to obtain high levels of soluble and active protein. The enzyme has been purified to near homogeneity. It appears a stable dimeric protein with a specific activity of 5500 units/mg protein, a subunit mass of 28 kDa and an isoelectric point of 9.0. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. Some kinetic properties of the recombinant protein have been compared with those of the promastigote enzyme and with the values previously reported for the T. brucei enzyme. The kinetics of the different enzyme preparations were very similar. For the recombinant enzyme the following values were measured: with glyceraldehyde 3-phosphate as substrate Km = 0.30 +/- 0.05 mM and kcat = 2.5 x 10(5) min-1; with dihydroxyacetone phosphate as substrate Km = 1.3 +/- 0.3 mM and kcat = 2.8 x 10(4) min-1.

Ultrastructural and Cytochemical Evidence for the Presence of Peroxisomes in the Generative Cell of Magnolia × soulangeana Pollen Grain

The generative cell (GC) development during three sequential stages of Magnolia × soulangeana pollen grain maturation was investigated by light and electron microscopy. Plastids were not identified in this cell but mitochondria, Golgi bodies and vesicles as well as rough endoplasmic reticulum profiles were always present. Microtubules were also present, their number increasing and their disposition varying during GC maturation. The most conspicuous components of the GC cytoplasm were the microbodies. The latter were few in number in the newly formed GC, and the appearance of their matrix was different from later developmental stages. A clear microbodial proliferation occurred in the GC during an intermediate stage of pollen maturation. Then, the microbody matrix was either fibrillar to granular as in the vegetative cell microbodies or very dense and compact. The polymorphism and size range and the frequent aggregation of these organelles in one or more clusters were also noteworthy. Tilting of semithin sections as well as the analysis of serial sections suggested that a number or enlarged and irregularly shaped microbodies co-exist with smaller and more spherical ones, the latter probably originating by budding. In the GC of the mature pollen the microbody-like organelles were in general more uniform both in shape and size. The cytochemical test of DAB was positive in the microbodies of both the pollen cells, thus demonstrating their peroxisomic nature. The function of the microbodies in the GC is not clear. In this cell, a few lipid droplets only exist during the first developmental stage and the microbodies were apparently unrelated to any other organelle. Possibly, these are unspecialized microbodies which are paternally transmitted, but it is not excluded that, temporarily, they may play some special role during GC maturation.

Cultivation of the sapropelic ciliate Plagiopyla nasuta Stein and isolation of the endosymbiont Methanobacterium formicicum

The sapropelic ciliate Plagiopyla nasuta was isolated and cultured in monoculture. Optimal conditions for growth were: 15–20°C, pH about 7, and about 2% of oxygen in the headspace. Cultures of P. nasuta produced methane. Epifluorescence microscopy revealed the presence of methanogenic bacteria as endosymbionts. An endosymbiont of the ciliate was isolated and identified as Methanobacterium formicicum. In the ciliate cell these methanogens were found to be closely associated with microbody-like organelles. No mitochondria could be detected.

Characterization of the genes for fructose-bisphosphate aldolase in Trypanosoma brucei

In Trypanosoma brucei stock 427 the glycolytic enzyme fructose-bisphosphate aldolase is encoded by two tandemly linked genes of identical sequence. Such a tandem arrangement of aldolase genes is also present in other T. brucei stocks of unrelated origin. In stock 427 one of the allelic genes is a pseudogene, as a result of a one-nucleotide deletion. The genes code for a polypeptide of 371 amino acids, with a calculated molecular weight of 40 940. The protein that is predicted from the gene sequence has 45–48% positional identity with known aldolase sequences of other organisms. The trypanosomal protein is, however, unique in having a 10 amino-acid insertion near its N-terminus and high number of basic residues, a feature it shares with other glycolytic enzymes of T. brucei. These glycolytic enzymes have in common that they are located in microbody-like organelles, the glycosomes. We have previously proposed that the positively charged residues may be involved in the import of the proteins into the organelles.

Ultrastructure of dormant basidiospores of Agaricus campestris

The basidiospore wall of Agaricus campestris Fr. consists of three distinct layers. The outer two layers are continuous around the spore, while the third layer originates only a short distance from the hilar appendage and quickly thickens to form the bulk of the wall material of the hilar appendage. The protoplast is surrounded by a typical plasma membrane which lacks distinct invaginations. Centrally located nonmembrane-bound lipid droplets comprise the bulk of the protoplasm. Spores are binucleate, but the two nuclei do not exhibit any distinct relationship to each other. Sausage-shaped mitochondria with only a few but well-delineated plate-like cristae are present. Scant endoplasmic reticulum occurs just beneath the plasma membrane. Ribosomes occur regularly attached to the endoplasmic reticulum and outer mitochondrial membrane, as well as being densely packed throughout the cytoplasm. The structure and possible functions of single membrane bound vacuoles and microbody-like organelles are discussed in relation to other basidiospores.

Ultrastructure and serial section reconstruction of zoospores of the fungus Phytophthora cinnamomi

Two zoospores of Phytophthora cinnamomi have been reconstructed from serial, 240-nm sections using high voltage electron microscopy. The mitochondrial complement is generally confined to the subperipheral zone of the cytoplasm and consists of 3 or 4 large, reticulate mitochondria and 19–23 small, unbranched mitochondria. In addition to peripheral cisternae and large peripheral vesicles, which also occur in the zoospores of related species, the peripheral cytoplasm of the P. cinnamomi zoopsores contains small, usually spherical vesicles. There are 105 and 121 large peripheral vesicles and 132 and 168 small peripheral vesicles in the two reconstructed cells. The morphology of the small peripheral vesicles is variable, with different forms showing evidence of regional localization. The cells also contain 45 and 84 endoplasmic reticulum-associated microbody-like organelles and 8–11 stacks of Golgi cisternae. These data, along with information on the structure and disposition of other organelles, provide a basis for ultrastructural comparisons of P. cinnamomi zoospores with those of other Oomycetes, and for studies of the structural and biochemical changes that occur during the early stages of infection of host plants.

Ultrastructure and Cytochemistry of Dormant Basidiospores ofPsilocybe Cubensis

The ultrastructure of dormant basidiospores of Psilocybe cubensis is described. The spore wall is characterized by three distinct layers and a germ pore. A pore cap is described for the first time in a species of Psilocybe. The protoplast is surrounded by a well defined plasma membrane with many distinct invaginations. Internally, large numbers of nonmembrane-bound lipids occur at both ends of the spore. Two nuclei are typically present and the nuclear envelope has many nuclear pores. Mitochondria with only a few, but well delineated, plate-like cristae are present. There is scant endoplasmic reticulum. Ribosomes occur regularly attached to the ER and outer mitochondrial membrane, as well as being densely packed throughout the cytoplasm. Variously sized vacuoles were demonstrated cytochemically to contain acid phosphatase. Microbody-like organelles are described but cytochemical tests to determine if these organelles are functionally similar to those of higher plants were unsuccessful. Cell-free extracts of basidiospores exhibit catalase and isocitrate lyase activity but no malate synthase

Hydrogenosomes in the rumen fungus Neocallimastix patriciarum.

Sedimentable hydrogenase activity was demonstrated in cell-free extracts from both zoospores and vegetative growth of the anaerobic rumen fungus Neocallimastix patriciarum. Electron micrographs of the fraction enriched in hydrogenase activity contained finely granular microbody-like organelles, about 0.5 micron in diameter and having an equilibrium density of about 1.2 g X ml-1 in sucrose, 1.12 g X ml-1 in Percoll and 1.27-1.28 g X ml-1 in Metrizamide. These organelles, which are sedimentable at 10(5) g-min, bear no similarity to mitochondria, but are morphologically similar to hydrogen-evolving organelles possessed by certain anaerobic protozoa and termed 'hydrogenosomes'. Other typical hydrogenosomal enzymes, namely 'malic' enzyme, pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin oxidoreductase, were enriched in the same particle fraction as hydrogenase. The synthesis of pyruvate:ferredoxin oxidoreductase was found to be suppressed when the organism was cultured under an atmosphere of CO2, and an alternative pathway is proposed for growth under these conditions.

Cross-linking of the enzymes in the glycosome of Trypanosoma brucei.

Glycosomes, the microbody-like organelles containing mainly glycolytic enzymes, were purified from the long slender bloodstream form of Trypanosoma brucei EATRO 110 monomorphic strain by an improved method in which the protozoa were frozen and thawed in 15% glycerol to free, from the plasma membrane, much of the variant surface glycoprotein which used to constitute the major contaminant of our purified glycosomes. The purified glycosomes have 11 major proteins, 6 of which, tentatively identified as phosphofructose kinase, hexokinase, 3-phosphoglycerate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alpha-glycerophosphate dehydrogenase, constitute 87% of the total glycosomal protein. The bifunctional cross-linking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate can penetrate the glycosomal membrane and cause extensive cross-linking of all the major glycosomal proteins. The cross-linked complex, insoluble in 0.1% Triton X-100 plus 0.15 M NaCl, contains all the glycosomal enzyme activities with only partial inactivations. All the enzymes are probably cross-linked into one large complex since they all sediment rapidly to the bottom of a 5-20% (v/v) sucrose density gradient. This successful cross-linking with reagents of span lengths of 11-12 A suggests close proximities among the glycosomal enzymes which may explain the extraordinarily high rate of glycolysis in T. brucei. Whether such a close association represents specific spatial arrangement required for genuine substrate channeling among the enzymes will be verified by future kinetic studies of the cross-linked enzyme complex.

Microbody-like organelles as taxonomic markers among oomycetes

Zoospores of Oomycetes contain a variety of microbody-like organelles with highly structured matrices. Although in general their function is unknown, the appearance of similar organelles in related taxa suggests the ultrastructural differences could be used as taxonomic characters. This study surveys microbody-like organelles of oomycetous zoospores to determine if this is an additional criterion by which the phylogeny of these fungi can be evaluated. In zoospores of the order Saprolegniales, kinetosome-associated organelles (K-bodies) are found which typically consist of tubular and/or granular matrices. K-bodies are not found associated with kinetosomes in zoospores of the Peronosporales, but microbodies containing tubules, and in some genera marginal plates, are located near the kinetosomes, along the groove, and in other peripheral areas. K-bodies have been reported in only one member of the order Lagenidiales. These K-bodies lack a granular matrix, but contain a single curved plate from which tubules arise, forming a cone. In the one genus of the Leptomitales examined, a similar K-body contains a plate and scattered tubules. Organisms with similar microbody-like organelles are probably more closely related than those with different types of microbody-like organelles. The presence of an organelle resembling K-bodies in zoospores of an alga in the Tribophyceae supports the phylogenetic association between algae and Oomycetes. A complete survey of Oomycete genera may well reveal intermediates between the structurally different types of microbody-like organelles, allowing the reconstruction of the phylogenetic history of an organelle.

Simultaneous purification of hexokinase, class-I fructose-bisphosphate aldolase, triosephosphate isomerase and phosphoglycerate kinase from Trypanosoma brucei.

A method is presented for the simultaneous purification of hexokinase, fructose-bisphosphate aldolase, triosephosphate isomerase and phosphoglycerate kinase, and the partial purification of glycerol-3-phosphate dehydrogenase (NAD+), 6-phosphofructokinase, glucosephosphate isomerase, and glycerol kinase from Trypanosoma brucei. As a first step, the glycosomes, microbody-like organelles of Trypanosomatidae, containing almost exclusively enzymes involved in glucose and glycerol metabolism [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360-364], were purified eightfold from homogenates with an average yield of 38%. Subsequently, the glycosomal content was subjected to hydrophobic interaction chromatography on phenyl-Sepharose. This step results in pure hexokinase (15% final yield) and almost pure triosephosphate isomerase, while the other glycosomal enzymes elute as mixtures of two or three enzymes. Triosephosphate isomerase was further purified to homogeneity on CM-cellulose (33% final yield), while phosphoglycerate kinase and fructose-bisphosphate aldolase were separated from each other and purified to homogeneity by affinity chromatography using ATP-Sepharose (25% and 30% final yields, respectively). Fructose-bisphosphate aldolase was further characterized as a typical class I enzyme.

Methanobacterium formicicum, an endosymbiont of the anaerobic ciliateMetopus striatus McMurrich

The Gram-positive methanogenic endosymbiont of the sapropelic ciliateMetopus striatus was isolated and identified asMethanobacterium formicicum. In the ciliate cell the methanogens are in close association with microbody-like organelles. No mitochondria could be detected. The nature of the microbodies and the physiological background of the observed association are discussed.

Hydrogenosomes in known species of rumen entodiniomorphid protozoa

The distribution of hydrogenosomal enzymes in several species of rumen entodiniomorphid protozoa grown in vivo and in vitro was investigated. Eudiplodinium maggii and Epidinium ecaudatum caudatum were shown to possess pyruvate synthase and hydrogenase enzyme activities associated with a fraction sedimentable at 105/ g/min and enriched in granular microbody-like organelles about 0.3 μm in diameter; these organelles are therefore morphologically and enzymically similar to hydrogenosomes from the rumen holotrichs. However, hydrogenase and pyruvate synthase were undetectable in several of the entodiniomorph species investigated.

Hydrogenosomes in known species of rumen entodiniomorphid protozoa

The distribution of hydrogenosomal enzymes in several species of rumen entodiniomorphid protozoa grown in vivo and in vitro was investigated. Eudiplodinium maggii and Epidinium ecaudatum caudatum were shown to possess pyruvate synthase and hydrogenase enzyme activities associated with a fraction sedimentable at 105/ g/min and enriched in granular microbody-like organelles about 0.3 μm in diameter; these organelles are therefore morphologically and enzymically similar to hydrogenosomes from the rumen holotrichs. However, hydrogenase and pyruvate synthase were undetectable in several of the entodiniomorph species investigated.

Hydrogenosomes in a mixed isolate of Isotricha prostoma and Isotricha intestinalis from ovine rumen contents

1. 1. Both Isotricha intestinalis and I. prostoma possess microbody-like organelles, with a highly granular appearance. 2. 2. These organelles, which are sedimentable at 10 5 g-min, bear no morphological similarity to mitochondria, but are enzymatically similar to organelles possessed by certain other anaerobic protozoa and termed hydrogenosomes. 3. 3. The hydrogenosomes isolated from a preparation of mixed isotrichs bear a closer similarity to those isolated from the other rumen holotrich, Dasytricha ruminantium, than those recently identified in a mixed entodiniomorph preparation, or the trichomonads, in that the enzyme malate dehydrogenase (decarboxylating) is non-sedimentable and phosphoacetyl transferase together with acetate kinase are involved in the transformation of acetyl-CoA to acetate. 4. 4. The results enable a scheme of acetate, CO 2 and H 2 formation from carbohydrates to be proposed and extends the number of protozoa known to possess this organelle.

Hydrogenosomes in the rumen protozoon Dasytricha ruminantium Schuberg.

This paper reports for the first time the presence in the anaerobic rumen ciliate Dasytricha ruminantium (Schuberg) of microbody-like organelles, about 0.5 micrometer diameter, with a granular matrix and an equilibrium density of approx. 1.18 g/ml. These organelles can be isolated in a fraction sedimented at 10(5) g-min that contains 67% of the total pyruvate synthase (EC 1.2.7.1), 66% of the hydrogenase (EC 1.18.3.1) and 20% of the lactate dehydrogenase (EC 1.1.1.27). Thus in several respects this fraction is enzymically similar to those containing hydrogenosomes in some other parasitic anaerobic protozoa (the trichomonads). However, in contrast with the hydrogenosomes of trichomonads, the oxygen-tolerant enzyme malate dehydrogenase (decarboxylating) (EC 1.1.1.40) is not particulate, but occurs only in the cytosol. These results enable the proposal of a scheme for the pathway of product formation (acetate, lactate, CO2 and H2) from carbohydrates.

Events surrounding the early development of Euglena chloroplasts : 18. Structure of the developing proplastid in the first hours of illumination from serial sections of wild-type cells

The structure of the proplastid in dark-grown nondividing cells of wild-type Euglena gracilis var. bacillaris and the changes which occur during the early hours of illumination have been studied using serial sections and transmission electron microscopy. The proplastid in dark-grown cells is a polyp-like structure with a number of tubular arms, each containing a noncrystalline prolamellar body composed of irregular tubules, at its extremity. The proplastid with membrane whorls attached is in intimate contact with the surrounding cytoplasm and is in close juxtaposition to microbody-like organelles and mitochondria. The plastid assumes a more globular shape after 30 min of illumination. During this period membrane whorls, similar to those described above, are found as transient structures at the interior of the prolamellar bodies and are found to be closely associated with mitochondria. Membrane whorls are also observed to penetrate to the cores of the microbodies. The thylakoids are attached to the remaining irregular tubular regions of the prolamellar body which surround the membrane whorls. There is a very close association of plastids, mitochondria, microbodies, and Golgi. After 1 to 2 hr the membrane whorls associated with the interior of the prolamellar bodies begin to disappear to be replaced by elongated straight tubules which, in turn, also disappear to leave a cavity surrounded by the remains of the original irregular tubules. There is a progressive pairing of thylakoids with the deposition of a dense matrix between the pairs; a different region of high density can be seen on either side of the paired thylakoids where single thylakoids adjoin them near the point of connection to the irregular tubules of the prolamellar bodies. No relations among the membrane whorls, straight tubules, and thylakoids have been detected. By 24 hr of development paired thylakoids are the rule. Reconstructions from serial sections are presented showing the three-dimensional structure of the proplastid before and after illumination.