Microbody Membrane Research Articles

Overproduction of a single protein, Pc-Pex11p, results in 2-fold enhanced penicillin production by Penicillium chrysogenum

Current industrial production of β-lactam antibiotics, using the filamentous fungus Penicillium chrysogenum, is the result of many years of strain improvement by classical mutagenesis. More efficient production strains showed significant increases in the number and volume fraction of microbodies in their cells, organelles that harbor key enzymes involved in the biosynthesis of β-lactam antibiotics. We have isolated the P. chrysogenum cDNA encoding Pc-Pex11p, a peroxin that is involved in microbody abundance. We demonstrate that overproduction of Pc-Pex11p in P. chrysogenum results in massive proliferation of tubular-shaped microbodies and a 2- to 2.5-fold increase in the level of penicillin in the culture medium. Notably, Pc-Pex11p-overproduction did not affect the levels of the enzymes of the penicillin biosynthetic pathway. Our results suggest that the stimulating effect of enhanced organelle numbers may reflect an increase in the fluxes of penicillin and/or its precursors across the now much enlarged microbody membrane.

Depletion of GIM5 Causes Cellular Fragility, a Decreased Glycosome Number, and Reduced Levels of Ether-linked Phospholipids in Trypanosomes

Microbody division in mammalian cells, trypanosomes, and yeast depends on the PEX11 microbody membrane proteins. The function of PEX11 is not understood, and the suggestion that it affects microbody (peroxisome) numbers in mammals and yeast, because it plays a role in beta-oxidation of fatty acids, is controversial. PEX11 and two PEX11-related proteins, GIM5A and GIM5B, are the predominant membrane proteins of the microbodies (glycosomes) of Trypanosoma brucei. The compartmentation of glycosomal enzymes is essential in trypanosomes. Deletion of the GIM5A gene from the form of the parasite that lives in the mammalian blood has no effect on trypanosome growth, but depletion of GIM5B on a gim5a null background causes death. We show here that procyclic trypanosomes, adapted for life in the Tsetse fly vector, survive without GIM5A and with very low levels of GIM5B. The depleted cells have fewer glycosomes than usual and are osmotically fragile, which is a novel observation for a microbody defect. Thus trypanosomes require both GIM5B and PEX11 for the maintenance of normal glycosome numbers. Procyclic cells lacking GIM5A, like mouse cells partially defective in PEX11, have fewer ether-linked phospholipids, even when GIM5B levels are not reduced. Metabolite measurements on GIM5A/B-depleted bloodstream form trypanosomes suggested a change in the flux through the glycolytic pathway. We conclude that PEX11 family proteins play important roles in determining microbody membrane structure, with secondary effects on a subset of microbody metabolic pathways.

Protein Transport in Plant Cells

The subcellular localization and secretion of proteins synthesized in the cytosol are determined by short amino acid sequences in their molecules. N-terminal transit peptides provide for protein translocation across the membranes of the ER, mitochondria, plastids, and microbodies. Later, these peptides are cleaved off by processing peptidases. C-terminal peptides direct some proteins into microbodies and vacuoles. Transport into the nucleus and insertion in the membranes are determined by the specific sequences that reside in the molecule of the mature protein. Specific receptors associated with the protein-translocating channel recognize transit peptides. Protein unfolding is required for successful protein transport through these channels. Chaperones maintain proteins in such a state. Folded proteins cross the nuclear pore complex and the membrane of microbodies. Protein transport is tightly associated with their processing. During the vesicular protein transport within the endomembrane system (ER, Golgi apparatus, plasma membrane, and vacuoles), correct protein targeting is ensured by protein sorting during vesicle loading, the assembly of corresponding protein coats, vesicle transport to the acceptor membrane, and specific membrane fusion.

An essential dimeric membrane protein of trypanosome glycosomes.

Kinetoplastid parasites compartmentalize the first seven enzymes of glycolysis in a peroxisome-like microbody, the glycosome. Genes encoding the most abundant protein of the glycosomal membrane, GIM5, have been cloned and the protein characterized. Two genes, GIM5A and GIM5B, encode 26 kDa proteins. Although many microbody membrane proteins are conserved in evolution, the only homologues of GIM5 in the available databases are from the closely related kinetoplastids Trypanosoma cruzi and Leishmania. The N- and C-termini are conserved between the two genes, and between species, and are oriented towards the cytosol. They are separated by a short loop that is located between two transmembrane domains and shows almost no sequence conservation. This suggests that the N- and C-terminal domains are more important for function. GIM5 forms dimers in vivo. Overexpression of GIM5B inhibits growth, whereas depletion of GIM5 to below 10% of wild-type levels is very rapidly lethal. This novel organellar membrane protein is therefore essential for bloodstream trypanosome survival.

Development of Microbody Membrane Proteins during the Transformation of Glyoxysomes to Leaf Peroxisomes in Pumpkin Cotyledons

Development of Microbody Membrane Proteins during the Transformation of Glyoxysomes to Leaf Peroxisomes in Pumpkin Cotyledons

Occurrence of peroxisomal membrane proteins in methylotrophic yeasts grown under different conditions

We have studied the substructure and polypeptide composition of the peroxisomal membranes in two methylotrophic yeasts in relation to different growth conditions. The results obtained indicated that no significant ultrastructural differences existed between the membranes of variously grown cells. The presence of specific peroxisomal membrane proteins (PMPs) was studied biochemically. On sodium dodecyl sulphate-polyacrylamide gels of purified microbody membranes isolated from methanol-grown Hansenula polymorpha, prominent protein bands were observed at 22, 31, 35, 42, 49 and 51 kD. These proteins were also present when the cells were grown in media containing ethanol and/or ethylamine. Apart from these, several other PMPs were specifically induced under these conditions, namely 24, 29, 37 and 62 kD proteins. The polypeptide composition of peroxisomal membranes from H. polymorpha was compared with that of another methylotroph, Candida biodinii. In the latter organism a specific PMP with a molecular weight of 23 kD was induced during growth on D-alanine instead of ammonium sulphate as the nitrogen source.

Electron cytochemical demonstration of phosphatase activity with microbody membranes of Basidiobolus haptosporus

Growth of cells of the potentially zoopathogenic fungus Basidiobolus haptosporus on a nutritionally defined medium with xanthine or urate as the nitrogen source results in greatly increased populations of microbodies. Modified Gomori procedures at the electron microscopic level suggested the single limiting membrane (and in some cases the granular matrix) of immature microbodies to be the exclusive subcellular locale(s) of alkaline phosphatase, 5'-nucleotidase and nucleoside diphosphatase activities. When grown in the presence of low inorganic phosphate, additional alkaline phosphatase activity was further identified cytochemically at and along profiles of endoplasmic reticulum and on inclusions previously described as "double-membraned vesicles". Cytochemical localization of acid phosphatase at microbody membranes was minimal if not ambiguous; Mg++-dependent adenosine triphosphatase and glucose-6-phosphatase were not identified at these locales. Quantitative biochemical estimates of alkaline phosphatase activity levels in particulate fractions initially increased with age of cells, perhaps as a function of the cultural induction and marked increase in immature microbody populations. We suggest that this enzyme may participate in some manner with protein translocation mechanisms associated with microbody biogenesis, ontogeny, and/or physiological function.

The peroxisome membrane: effects of detergents and lipid solvents upon its ultrastructure and permeability to catalase

The peroxisome (microbody) membrane differs markedly from other cytoplasmic membranes, especially the lysosomal, in its susceptibility to various disruptive treatments and detergents. Although this phenomenon has been extensively studied in biochemical literature, there have been no ultrastructural or cytochemical investigations of it. We now report on the effects of detergents, organic lipid solvents, and several adjuvants used in cell fractionation upon the ultrastructure of the peroxisomal membrane and its permeability to catalase, as demonstrated cytochemically by the DAB reaction and biochemically by the assay of catalase activity.

The peroxisome (microbody) membrane: effects of detergents and lipid solvents on its ultrastructure and permeability to catalase.

The effects of detergents, organic lipid solvents, and several adjuvants used in cell fractionation on the ultrastructure of the peroxisomal (microbody) membrane and its permeability to catalase have been investigated. Chopper sections of glutaraldehyde-fixed liver were incubated in the presence of various agents, followed by cytochemical staining for catalase and processed for electron microscopy. Catalase activity was also determined biochemically in the incubation medium. Marked catalase diffusion was found after treatment with 1% or 0.5% Triton X-100 or deoxycholate, as well as with 50% ethanol or acetone or 20% propanol or t-butanol. In contrast, 1% digitonin and lower concentrations of the above agents, as well as sucrose or glycerine caused selective diffusion of catalase from a limited population of peroxisomes. Treatment with 10% polyvinylpyrrolidone (PVP), which has been used as a protective agent in the isolation of microbodies, did not produce any alteration in the fine structure and cytochemical appearance of peroxisomes. These findings concur with earlier biochemical studies on freshly isolated peroxisomes and demonstrate the susceptibility of microbodies, even in glutaraldehyde-fixed rat liver to the effects of various agents which affect the microbody membrane. A close correlation between the ultrastructural integrity of the microbody membrane and its permeability to catalase has been found. The significance of these observations for the assessment of the permeability characteristics of the microbody membrane is discussed.

Peroxisomes: Identification in freeze-etch preparations of rat kidney

The definitive identification of peroxisomes in freeze-etch preparations of normal mammalian cells has been impeded due to their similarity to other globular cytoplasmic organelles, especially the lysosomes. In the proximal tubules of the rat kidney there is a distinct group of large microbodies with an angular shape and crystalline inclusions which have now been identified in freeze-etch replicas. Rat kidneys were fixed by perfusion with glutaraldehyde and processed for catalase cytochemistry and freeze-etching. In ultrathin sections catalase positive peroxisomes 0.5 – 2 μm in diameter with an angular multifaceted shape and scalloped margins were found. In freeze-etch replicas particles with the same size, shape, and intracellular distribution were found; these contained branching and anastomosing tubular inclusions. The tubules, 100 – 125 nm in diameter, left deep impressions upon the limiting membrane of peroxisomes. A crystalline pattern with a periodicity of approximately 92 ± 8 Å perpendicular to the axis of the tubules was noted on the E-face of the microbody membrane. In addition, a few membrane particles were present on the E-face, but many more particles were noted on the Pface of the microbody membrane. The cisternae of ER, with typical fenestrations, wrapped around large portions of the surface of peroxisomes, thus demonstrating the close association of these two organelles.

Association of microbodies, Woronin bodies, and septa in intercellular hyphae of Cymadothea trifolii

This ultrastructural study describes the origin of Woronin bodies from microbodies in intercellular hyphae of the plant pathogen Cymadothea trifolii. In this fungus the development of Woronin bodies begins with the appearance of electron-dense material within the microbody. Subsequently, this material is confined to a developing evagination of the delimiting microbody membrane. Eventually the protrusion containing the electron-dense material is released as a Woronin body via an exocytotic mechanism. Woronin bodies are frequently found near septal pores and probably contribute to the formation of septal pore plugs.

Morphology and ontogeny of microbodies in the oomycete fungus Sapromyces elongatus

Ultrastructural observations on microbodies in hyphae and sporangia of the oomycete fungus Sapromyces elongatus reveal that they consistently develop adjacent to cisternae of endoplasmic reticulum. At maturity they contain a crystalline structure, having a tetragonal lattice with asymmetric spacings of 12 and 13 nm and also several small irregular tubular structures. Mature microbodies also develop numerous villous invaginations, each filled with a cistema of endoplasmic reticulum, the membrane of which is constantly spaced from, and possibly cross bridged to, the microbody membrane. Although microbodies may be appressed to mitochondria, they do not show any associations with lipid droplets. In apparent senescence the crystals remain, but the microbody membrane enlarges greatly with accompanying loss of associated endoplasmic reticulum and dilution of matrix, so that the last recognizable stage is a crystal-containing vacuole. Zoospores contain numerous mature, invaginated microbodies associated with cisternae of endoplasmic reticulum; they are clustered predominantly around the nucleus. The morphology of the microbodies and their possession of crystalline inclusions differ from previously reported oomycete microbodies and may have taxonomic value, whereas the postulated ontogenic sequence and fate of the microbodies are previously unreported and may represent a general mechanism for microbody breakdown.

Plant microbody proteins, III. Labelling of the peroxisomal membrane protein SP-63 in vitro and in vivo.

Methods were developed to charcterize membranes and membrane components of leaf peroxisomes from Lens culinaris. While microbodies from etiolated young leaves exhibited an equilibrium density of 1.19 g/cm3, older leaves or leaves exposed to light for increasing periods of time contained microbodies banding at higher densities up to 1.235 g/cm3. Similar values were also found for the corresponding microbody membranes, which could be labelled with diazotized [35S]sulphanilic acid. Labelling was also performed using proteins extracted from the membranes. Their main structural protein (SP-63) was solubilized with sodium dodecylsulphate and labelled with fluorescent compounds as well as with diazotized [35S]sulphanilic acid or [3H]iodoacetic acid. These conversions greatly facilitate all analytical procedures, e.g. tracing the migration of SP-63 in gels or the movement in sucrose gradients containing sodium dodecylsulphate during zonal centrifugation. Also, labelling of SP-63 in vivo was accomplished when labelled amino acids were infused into etiolated leaves while exposing them to light.

Density-labeling evidence against a de novo formation of peroxisomes during greening of fat-storing cotyledons

During greening of fat-storing cotyledons, the functional properties of the microbody population changes from that of glyoxysomes to that of leaf peroxisomes. The amount of microbody protein per cell is assumed to be substantially reduced [ 1,2,3] . Little is known about the mechansim of this change of microbody function. Recently, data obtained from labeling of microbody membranes with Cl4 and H3 choline were interpreted as evidence that light triggers both a selective destruction of glyoxysomes and a concomitant de novo formation of leaf peroxisomes [4]. In contrast, electron microscopic examinations [.5,6] as well as studies on the change of microbody isoenzyme spectra [2,7] had previously led to the conclusion that in greening cotyledons leaf peroxisomes do arise from glyoxysomes. Direct evidence for a de novo synthesis of proteins is provided by density labeling with stable heavy isotopes [8]. In this paper the lack of light-dependent density labeling of leaf peroxisomes is contrasted with light-dependent density labeling of plastids, and with density labeling of glyoxysomes in early stages of germination of sunflower seeds.

The effect of lipid extraction on electron-microscopic images of plant membranes

An evaluation was made of the role of lipids in electron-microscopic membrane images of plant cells by comparing extracted lipids with changes in the ultrastructural membrane images. Lipids were extracted from tobacco leaves with a series of acetone concentrations. In a parallel series, glutaraldehyde fixation preceded lipid extraction. Thin-layer chromatography of the acetone extracts showed no major difference in the lipids extracted with and without glutaraldehyde fixation, but different concentrations of acetone removed specific lipids. Electron micrographs of tissues not previously fixed with glutaraldehyde showed a disruption of all membrane images at acetone concentrations greater than 30%. From these studies it appears that lipids are involved in the formation of electron-microscopic membrane images, but to a different degree in the various membranes. The general form of mitochondria and chloroplast grana was not dependent upon lipid, though lipid was required for the typical density pattern of the granal partitions. The bounding membranes of the mitochondria and chloroplasts were lost with extraction of galactolipids, sulfolipids, and phospholipids. The plasmalemma, tonoplast, and microbody membranes lost their typical density pattern and their structural integrity with the extraction of phospholipids.

Ultrastructural localization of glutamic oxaloacetic transaminase (GOT) activity in hamster tissues.

Ultrastructural localization of the aldehyde resistant glutamic oxaloacetic transaminase (GOT) activity was studied in various organs of the hamster. Mitochondrial GOT activity was located in the intracristal space and, to a lesser extent, in the outer compartment, mostly on the surface of the inner membrane. There was very little evidence of reaction directly over the membrane or in the matrix of the mitochondria. The mitochondria of the cardiac and the skeletal muscle showed the highest level of activity both in the outer compartment and in the intracristal space. The cells in the liver and adrenal cortex had considerable amount of patchy reaction praducts. In both the axonal portion of the motor endplate and in neurons of the cerebrum, this enzyme activity was found mainly in the intracristal space of mitochondria. The mitochondria in the smooth muscle, the kidney, and the intestine showed a lower level of activity. Occasionaly the limiting membrane of microbodies in the hepatocytes showed GOT activity. No reaction product was seen on the smooth surfaced membrane systems such as the Golgi complex, the agranular endoplasmic reticulum and the plasma membrane.

A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue

Microbodies (peroxisomes and glyoxysomes), mitochondria, and microsomes from rat liver, dog kidney, spinach leaves sunflower cotyledons, and castor bean endosperm were isolated by sucrose density-gradient centrifugation. The microbody-limiting membrane and microsomes each contained NADH-cytochrome c reductase and had a similar phospholipid composition. NADH-cytochrome c reductase from plant and animal microbodies and microsomes was insensitive to antimycin A, which inhibited the activity in the mitochondrial fractions. The pH optima of cytochrome c reductase in plant microbodies and microsomes was 7.5–9.0, which was 2 pH units higher than the optima for the mitochondrial form of the enzyme. The activity in animal organelles exhibited a broad pH optimum between pH 6 and 9. Rat liver peroxisomes retained cytochrome c reductase activity, when diluted with water, KCl, or EDTA solutions and reisolated. Cytochrome c reductase activity of microbodies was lost upon disruption by digitonin or Triton X-100, but other peroxisomal enzymes of the matrix were not destroyed. The microbody fraction from each tissue also contained a small amount of NADH-cytochrome b 5 reductase activity. Peroxisomes from spinach leaves were broken by osmotic shock and particles from rat liver by diluting in alkaline pyrophosphate. Upon recentrifugation liver peroxisomes yielded a core fraction containing urate oxidase at a sucrose gradient density of 1.23 g × cm −3, a membrane fraction at 1.17 g × cm −3 containing NADH-cytochrome c reductase, and soluble matrix enzymes at the top of the gradient. Relative to other organelles the microbodies had a very low level of phospholipid (0.03–0.09 mg per mg protein). Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and phosphatidyl serine were found in all organelle fractions. The percentage of each phospholipid in isolated microbodies from rat liver or spinach leaves was similar in the microbodies and microsomes. Glyoxysomes from germinating castor beans contained little phosphatidyl serine but rather an unidentified phospholipid. From spinach leaves the microsomes contained much phosphatidyl serine and the mitochondria had a significant amount of cardiolipin.

Charakterisierung der Microbodies aus Spadix-Appendices von Arum maculatum L. und Sauromatum guttatum Schott

When the sections of the spadix appendix of Arum are incubated in a medium containing diaminobenzidine and H2O2, only the membrane of microbodies is stained. On the other hand, microbodies of Sauromatum show a stained matrix as usual. Catalase-containing cell organelles isolated from spadix appendices of Arum show the same typical membrane staining as the microbodies in situ do. Thus the identity of these organelles with microbodies seems to be proved. After anthesis the microbodies in situ usually do not give a positive reaction for catalase with diaminobenzidine and H2O2. However, cytochemical and biochemical tests for catalase on microbodies isolated during this stage of development clearly demonstrate the presence of this enzyme. Uricase is localized in the microbodies of Arum as well as catalase. No malate dehydrogenase, peroxidase, and allantoinase could be found in the microbodies. Before anthesis the microbodies of spadix appendices of Arum have an equilibrium density in aqueous sucrose of 1.22 gcm-3. After anthesis the density changes into 1.23 to 1.24 gcm-3.

Ultrastructural changes in tobacco undergoing the hypersensitive reaction caused by plant pathogenic bacteria

The hypersensitive reaction induced by either Erwinia amylovora or Pseudomonas pisi is indicative of widespread damage to membranes of subcellular organelles and particulate cellular components in tobacco leaf tissue. Plasmalemma, tonoplast, as well as the bounding and internal membranes of chloroplasts and mitochondria and the external membrane of microbodies, are profoundly deranged in 7 h. Cytoplasm, groundplasm of chloroplasts, mitochondria and microbodies and membrane-attached and free ribosomes are similarly affected. The damage coincides with and is probably the cause of the observed tissue collapse and rapid loss of electrolyte-laden water. The presence of few bacterial cells (not more than 4 × 10 6 cells/leaf disk 1 cm in diameter) cause the observed symptoms which appear, at the ultrastructural level, to be unique.

The widespread occurrence of plant cytosomes resembling animal microbodies

Single membrane bounded organelles characterized by a physical association with endoplasmic reticulum have been observed in a wide range of cell types and plant species including Gymnosperm, Angiosperm, Pteridophyte, and Thallophyte (algae and fungi) tissues. The morphological similarity between these organelles and animal microbodies suggests that they are cytological homologues. Plant microbodies were observed both with and without dense internal inclusions but unlike animal microbodies could not be shown to contain uricase. Plant microbody membranes are resistant to degenerative influences and remain associated with a small portion of endoplasmic reticulum even in isolated cell fractions.