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W Harder

Publications and source records attributed to W Harder.

At least 19 recordsLinked to original sources
PubMed1992

Hansenula polymorpha: an attractive model organism for molecular studies of peroxisome biogenesis and function.

In wild-type Hansenula polymorpha the proliferation of peroxisomes in induced by various unconventional carbon- and nitrogen sources. Highest induction levels, up to 80% of the cytoplasmic volume, are observed in cells grown in methanol-limited chemostat cultures. Based on our accumulated experience, we are now able to precisely adjust both the level of the peroxisome induction as well as their protein composition by specific adaptations in growth conditions. During the last few years a series of "peroxisome-deficient (per) mutants of H. polymorpha have been isolated and characterized. Phenotypically these mutants are characterized by the fact that they are not able to grow on methanol. Three mutant phenotypes were defined on the basis of morphological criteria, namely: (a) mutants completely lacking peroxisomes (Per-;13 complementation groups); (b) mutants containing few small peroxisomes which are partly impaired in the peroxisomal import of matrix proteins (Pim-; five complementation groups); and (c) mutants with aberrations in the peroxisomal substructure (Pss-; two complementation groups). In addition, several conditional Per-, Pim- and Pss- mutants have been obtained. In all cases the mutant phenotype was shown to be caused by a recessive mutation in one gene. However, we observed that different mutations in one gene may cause different morphological mutant phenotypes. A detailed genetic analysis revealed that several PER genes, essential for peroxisome biogenesis, are tightly linked and organized in a hierarchical fashion. The use of both constitual and conditional per mutants in current and future studies of the molecular mechanisms controlling peroxisome biogenesis and function is discussed.

Fungal Proteins↗
PubMed1992

Peroxisome biogenesis in Hansenula polymorpha: different mutations in genes, essential for peroxisome biogenesis, cause different peroxisomal mutant phenotypes.

In Hansenula polymorpha, different monogenic recessive mutations mapped in either of two previously identified genes, PER1 and PER3, produced different peroxisomal mutant phenotypes. Among five per1 mutants, four showed a Pim- phenotype: the cells contained few small peroxisomes while the bulk of the matrix enzymes resided in the cytosol. One of these mutants, per1-124 had an enhanced rate of peroxisome proliferation. The fifth mutant completely lacked peroxisomes (Per- phenotype). Of seven per3 mutants, four displayed a Pim- phenotype, two others a Per- phenotype, while one mutant showed pH-dependent growth on methanol and was affected in oligomerization of peroxisomal matrix protein. Thus, the protein products of both PER1 and PER3 genes appear to be essential in different aspects of peroxisome assembly/proliferation.

Genes, Fungal↗
PubMed1992

Formation and quantification of protein complexes between peroxisomal alcohol oxidase and GroEL.

We have studied the use of yeast peroxisomal alcohol oxidase (AO) as a model protein for in vitro binding by GroEL. Dilution of denatured AO in neutral buffer leads to aggregation of the protein, which is prevented by the addition of GroEL. Formation of complexes between GroEL and denatured AO was demonstrated by a gel-shift assay using non-denaturing polyacrylamide gel electrophoresis, and quantified by laser-densitometry of the gels. In the presence of MgAMP-PNP or MgADP the affinity of GroEL for AO was enhanced. Under these conditions up to 70% of the purified GroEL formed a complex with this protein. Release was stimulated at room temperature by MgATP, and was further enhanced by addition of GroES.

Alcohol Oxidoreductases↗
PubMed1992

Location of catalase in crystalline peroxisomes of methanol-grown Hansenula polymorpha.

We have studied the intraperoxisomal location of catalase in peroxisomes of methanol-grown Hansenula polymorpha by (immuno)cytochemical means. In completely crystalline peroxisomes, in which the crystalline matrix is composed of octameric alcohol oxidase (AO) molecules, most of the catalase protein is located in a narrow zone between the crystalloid and the peroxisomal membrane. In non-crystalline organelles the enzyme was present throughout the peroxisomal matrix. Other peroxisomal matrix enzymes studied for comparison, namely dihydroxyacetone synthase, amine oxidase and malate synthase, all were present throughout the AO crystalloid. The advantage of location of catalase at the edges of the AO crystalloids for growth of the organism on methanol is discussed.

Alcohol Oxidoreductases↗
PubMed1992

Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha.

Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an H. polymorpha replicon-like sequence present on the S. cerevisiae LEU2 gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multi-copy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in H. polymorpha. In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.

DNA Repair↗
PubMed1992

Unique and overlapping pollutant stress proteins of Escherichia coli.

Exposure of growing batch cultures of Escherichia coli to nine different "model micropollutants" (benzene, cadmium chloride, chlorpyrivos, 2,4-dichloroaniline, dioctylphtalate, hexachlorobenzene, pentachlorophenol, trichloroethylene, and tetrapropylbenzosulfonate) led to the induction of 13 to 39 proteins, as analyzed by two-dimensional gel electrophoresis. Some of these proteins overlapped with heat shock and carbon starvation proteins, but at least 50% were unique to a given chemical. The stress protein induction showed a temporal pattern, indicating sequential gene expression. Chemical stress protein synthesis occurred even at concentrations that had no effect on growth. Thus, the synthesis of these proteins can be a sensitive index of stress and the nature of environmental pollution.

Bacterial Proteins↗
PubMed1992

Development of multipurpose peroxisomes in Candida boidinii grown in oleic acid-methanol limited continuous cultures.

We have studied the development and metabolic significance of peroxisomes in the yeast Candida boidinii following adaptation of the organism to cultivation conditions which require the simultaneous presence and activity of two independent peroxisome-mediated pathways for growth. After the addition of methanol to oleic acid-grown cells at late exponentional growth, a number of new small peroxisomes developed which, apart from the presence of beta-oxidation enzymes, were characterized by the presence of enzymes involved in methanol metabolism (alcohol oxidase and dihydroxyacetone synthase). The latter proteins, however, were absent in the larger organelles which were originally present in the oleic acid-grown cells prior to the addition of methanol and which contained only enzymes of the beta-oxidation pathway. Subsequent experiments on cells from continuous cultures grown on a mixture of oleic acid and methanol at steady-state conditions revealed that both the enzymes of the beta-oxidation pathway and those involved in methanol metabolism were found in one and the same compartment. Thus, under these conditions the cells contained peroxisomes which were concurrently involved in the metabolism of two different carbon sources simultaneously used for growth. Our results indicated that the heterogeneity in the peroxisomal population of a single cell, observed in the transient state following the addition of methanol, is only temporary and due to heterogeneity among these organelles with respect to their capacity to incorporate newly synthesized matrix proteins.

Acetyl-CoA C-Acetyltransferase↗
PubMed1991

Physiological studies on the utilization of oleic acid by Saccharomyces cerevisiae in relation to microbody development.

We studied the influence of specific growth conditions on the induction of beta-oxidation enzymes and rate of microbody proliferation in S. cerevisiae by oleic acid. Of all conditions tested, highest enzyme levels and microbody numbers were achieved in glucose-limited continuous cultures, supplemented with oleic acids second carbon source. Comparable enzyme levels were observed in identical cultures of peroxisome-deficient (pas) mutants of S. cerevisiae. These experiments showed chemostat cultures on glucose/oleic acid mixtures to be a method of choice for future studies on microbody biogenesis/assembly in constructed, conditional pas mutants.

Cell Division↗
PubMed1991

Purification and characterization of an activator protein for methanol dehydrogenase from thermotolerant Bacillus spp.

All thermotolerant methanol-utilizing Bacillus spp. investigated by us possess a NAD-dependent methanol dehydrogenase (MDH) activity which is stimulated by a protein present in the soluble fraction of Bacillus sp. C1 cells. This activator protein was purified to homogeneity from Bacillus sp. C1 cells grown at a low dilution rate in a methanol-limited chemostat culture. The native activator protein (Mr = 50,000) is a dimer of Mr = 27,000 subunits. The N-terminal amino acid sequence revealed no significant similarity with any published sequences. Stimulation of MDH activity by the activator protein required the presence of Mg2+ ions. Plots of specific MDH activity versus activator protein concentration revealed Michaelis-Menten type kinetics. In the presence of activator protein, MDH displayed biphasic kinetics (v versus substrate concentration) toward C1-C4 primary alcohols and NAD. The data suggest that in the presence of activator protein plus Mg2+ ions, MDH possesses a high affinity active site for alcohols and NAD, in addition to an activator- and Mg2(+)-independent low affinity active site. The activation mechanism remains to be elucidated.

Alcohol Oxidoreductases↗
PubMed1991

Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeasts: a review.

Alcohol oxidase (AO) catalyses the first step of methanol metabolism in yeasts. In vivo the enzyme is compartmentalized in special cell compartments, called peroxisomes. The enzyme along with the organelles are induced during growth of methylotrophic yeasts on methanol as the sole carbon source. Like all other peroxisomal matrix proteins, AO is encoded by a nuclear gene. Expression of the protein is regulated by a repression/derepression mechanism, but also by induction. Inactive monomeric precursor protein is synthesized in the cytosol and subsequently imported post-translationally into peroxisomes without further processing. Assembly into the active homo-octameric enzyme and binding of the prosthetic group flavin adenine dinucleotide occurs inside the organelle. When enhanced concentration of octameric alcohol oxidase are present in the organelles, the enzyme may form a crystalloid. Oligomerization is not dependent on translocation of AO precursors into their target organelle since octameric, active AO is detected in the cytosol and nucleus of peroxisome-deficient mutants of Hansenula polymorpha: at high expression rates large cytosolic AO crystalloids are formed, which occasionally are also encountered inside the nucleus of such mutants. This paper summarizes recent findings and views on the mechanisms involved in synthesis, import, assembly and crystallization of this important peroxisomal enzyme.

Alcohol Oxidoreductases↗
PubMed1991

Selective inactivation of alcohol oxidase in two peroxisome-deficient mutants of the yeast Hansenula polymorpha.

We have studied selective inactivation of alcohol oxidase (AO) in two peroxisome-deficient (PER) mutants of the yeast Hansenula polymorpha. In these mutants high activities of cytosolic AO are induced by different growth conditions. At enhanced expression rates AO is arranged in large crystalloids in the cytosol, whereas smaller crystalloids are often observed inside the nucleus. Transfer of cells of the PER mutant 125-2E, which completely lacks peroxisomes, to glucose-excess conditions did not lead to degradative inactivation of AO and catalase as observed in wild-type (WT) cells used as a control. The gradual decrease in enzyme activities in the PER mutant could be accounted for by dilution of existing enzyme into newly formed cells as a result of growth. Morphologically, degradation of the cytosolic crystalloids was also not observed. Similar results were obtained with a second PER mutant (strain 124-2D), impaired in the import of peroxisomal matrix proteins. This mutant is characterized by the presence of small peroxisomes and large cytosolic AO crystalloids. Upon a shift of cells to glucose-excess conditions only part of the small peroxisomes present in these cells were degraded by mechanisms similar to those observed in WT cells placed under identical conditions. These results indicate that degradative inactivation of AO in H. polymorpha is strictly dependent on the localization of the enzyme inside peroxisomes and furthermore suggests that the mechanisms triggering this process are not directed against AO protein, but instead, to the membrane surrounding the organelle. Transfer of cells to methanol- or ethanol-containing media both resulted in modification inactivation of AO.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗
PubMed1991

Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha: a physiological study.

We have studied methanol-utilization in a peroxisome-deficient (PER) mutant of Hansenula polymorpha. In spite of the fact that in carbon-limited chemostat cultures under induced conditions the enzymes involved in methanol metabolism were present at wild-type (WT) levels, this mutant is unable to grow on methanol as a sole carbon and energy source. Addition of methanol to glucose-limited (SR = 12.5 mM) chemostat cultures of the PER mutant only resulted in an increase in yield when small amounts were used (up to 22.5 mM). At increasing amounts however, a gradual decrease in cell density was observed which, at 80 mM methanol in the feed, had dropped below the original value of the glucose-limited culture. This reduction in yield was not observed when increasing amounts of formate instead of methanol were used as supplements for the glucose-limited mutant culture and also not in WT cells, used as control in these experiments. The effect of addition of methanol to a glucose-limited PER culture was also studied in the transient state during adaptation of the cells to methanol. The enzyme patterns obtained suggested that the ultimate decrease in yield observed at enhanced methanol concentrations was due to an inefficient methanol-metabolism as a consequence of the absence of peroxisomes. The absence of intact peroxisomes results in two major problems namely i) in H2O2-metabolism, which most probably is no longer mediated by catalase and ii) the inability of the cell to control the fluxes of formaldehyde, generated from methanol. The energetic consequences of this metabolism, compared to the WT situation with intact peroxisomes, are discussed.

Alcohol Dehydrogenase↗
PubMed1991

Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha.

Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95%, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in Vmax and a significant (about one order) decrease in the Km of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k = 0.78 h-1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (kapp = 0.27 h-1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as D-amino acid oxidase from Candida boidinii.

Alcohol Oxidoreductases↗
PubMed1990

Immunocytochemical evidence for the acidic nature of peroxisomes in methylotrophic yeasts.

The possible acidic nature of the peroxisomal matrix present in intact yeast cells was studied immunocytochemically, using the weak base DAMP as a probe. Spheroplasts of methanol-grown Candida boidinii and Hansenula polymorpha were regenerated and incubated with DAMP. After immunogold labelling, using antibodies against DAMP, a specific accumulation of gold particles was observed on the peroxisomal profiles. This labelling was absent in controls, performed in the presence of ionophores or chloroquine. These results support earlier observations, that in intact cells a pH-gradient exists across the peroxisomal membrane. Experiments, carried out on osmotically swollen spheroplasts indicated that maintenance of this pH-gradient is strongly related to the cell's integrity.

Candida↗
PubMed1990

Permeability properties of peroxisomal membranes from yeasts.

We have studied the permeability properties of intact peroxisomes and purified peroxisomal membranes from two methylotrophic yeasts. After incorporation of sucrose and dextran in proteoliposomes composed of asolectin and peroxisomal membranes isolated from the yeasts Hansenula polymorpha and Candida boidinii a selective leakage of sucrose occurred indicating that the peroxisomal membranes were permeable to small molecules. Since the permeability of yeast peroxisomal membranes in vitro may be due to the isolation procedure employed, the osmotic stability of peroxisomes was tested during incubations of intact protoplasts in hypotonic media. Mild osmotic swelling of the protoplasts also resulted in swelling of the peroxisomes present in these cells but not in a release of their matrix proteins. The latter was only observed when the integrity of the cells was disturbed due to disruption of the cell membrane during further lowering of the concentration of the osmotic stabilizer. Stability tests with purified peroxisomes indicated that this leak of matrix proteins was not associated with the permeability to sucrose. Various attempts to mimic the in vivo situation and generate a proton motive force across the peroxisomal membranes in order to influence the permeability properties failed. Two different proton pumps were used for this purpose namely bacteriorhodopsin (BR) and reaction center-light-harvesting complex I (RCLH1 complex). After introduction of BR into the membrane of intact peroxisomes generation of a pH-gradient was not or barely detectable. Since this pump readily generated a pH-gradient in pure liposomes, these results strengthened the initial observations on the leakiness of the peroxisomal membrane fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Candida↗