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W Harder

Publications and source records attributed to W Harder.

At least 37 records · Page 2Linked to original sources
PubMed1994

Assembly of alcohol oxidase in peroxisomes of the yeast Hansenula polymorpha requires the cofactor flavin adenine dinucleotide.

The peroxisomal flavoprotein alcohol oxidase (AO) is an octamer (600 kDa) consisting of eight identical subunits, each of which contains one flavin adenine dinucleotide molecule as a cofactor. Studies on a riboflavin (Rf) auxotrophic mutant of the yeast Hansenula polymorpha revealed that limitation of the cofactor led to drastic effects on AO import and assembly as well as peroxisome proliferation. Compared to wild-type control cells Rf-limitation led to 1) reduced levels of AO protein, 2) reduced levels of correctly assembled and activated AO inside peroxisomes, 3) a partial inhibition of peroxisomal protein import, leading to the accumulation of precursors of matrix proteins in the cytosol, and 4) a significant increase in peroxisome number. We argue that the inhibition of import may result from the saturation of a peroxisomal molecular chaperone under conditions that normal assembly of a major matrix protein inside the target organelle is prevented.

Alcohol Oxidoreductases↗
PubMed1993

Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenula polymorpha.

We have studied the fate of the watermelon (Citrullus vulgaris Schrad.) glyoxysomal enzyme, malate dehydrogenase (gMDH), after synthesis in the methylotrophic yeast, Hansenula polymorpha. The gene encoding the precursor form of gMDH (pre-gMDH) was cloned in an H. polymorpha expression vector downstream of the inducible H. polymorpha alcohol oxidase promoter. During methylotrophic growth, pre-gMDH was synthesized and imported into peroxisomes, where it was enzymatically active. The apparent molecular mass of the protein located in H. polymorpha peroxisomes was equal to that of pre-gMDH (41 kDa), indicating that N-terminal processing of the transit peptide had not occurred in the yeast.

Blotting, Western↗
PubMed1993

Peroxisome biogenesis in the yeast Hansenula polymorpha is controlled by a complex set of interacting gene products.

We have studied the genetic interactions between mutant alleles in 12 genes, designated PER1-PER12, which are essential for peroxisome biogenesis in the yeast Hansenula polymorpha. Recessive mutations in any of these genes determined three different morphological phenotypes: (i) complete absence of peroxisomes (Per-); (ii) presence of small peroxisomes in conjunction with a major fraction of peroxisomal matrix proteins in the cytosol (Pim-); and (iii) presence of peroxisomes with aberrant crystalline matrix substructure (Pss-). Extensive complementation analysis showed many cases of noncomplementation--that is, diploids that contained both wild-type and mutant alleles of two different PER genes were unable to grow on methanol and showed peroxisomal defects. The observed cases of unlinked noncomplementation appeared to be gene and allele specific and were predominantly observed at lower temperatures (cold sensitive). The genetic results obtained were used to formulate a model of PER gene product interactions. In this model, five PER gene products are key or core components of the complex. Other PER gene products appear to play a more peripheral role.

Alleles↗
PubMed1993

Transient repression of the synthesis of OmpF and aspartate transcarbamoylase in Escherichia coli K12 as a response to pollutant stress.

The synthesis of total cellular proteins in Escherichia coli K12 was studied in batch culture following exposure of cells to low concentrations of monochlorophenol, pentachlorophenol and cadmium chloride. Changes in protein patterns were identified after pulse-chase labelling of proteins with [35S]methionine and subsequent two-dimensional gel electrophoresis (2D-PAGE). We demonstrated that besides the induction of some stress proteins, also a transient decrease in the rate of synthesis of other proteins occurred. Two of these proteins were identified as OmpF and aspartate transcarbamoylase (ATCase). Their transient repression appeared to be a general response to stress elicited by different pollutants and may therefore be used as a general and sensitive early warning system for pollutant stress.

Aspartate Carbamoyltransferase↗
PubMed1993

Affinity purification of molecular chaperones of the yeast Hansenula polymorpha using immobilized denatured alcohol oxidase.

We used peroxisomal alcohol oxidase (AO) for the affinity purification of molecular chaperones from yeasts. Methodical studies showed that up to 0.8 mg of purified bacterial GroEL was able to bind per ml of immobilized denatured AO column material. Using crude extracts of Hansenula polymorpha or Saccharomyces cerevisiae, several proteins were specifically eluted with Mg-ATP which were recognized by antibodies against hsp60 or hsp70. One H. polymorpha 70 kDa protein was strongly induced during growth at elevated temperatures, whereas a second 70 kDa protein as well as a 60 kDa protein showed strong protein sequence homology to mitochondrial SSC1 and hsp60, respectively, from S. cerevisiae.

Alcohol Oxidoreductases↗
PubMed1993

Expression and targeting of a 47 kDa integral peroxisomal membrane protein of Candida boidinii in wild type and a peroxisome-deficient mutant of Hansenula polymorpha.

A 47 kDa integral peroxisomal membrane protein (PMP47) of Candida boidinii was expressed in wild type (WT) and a temperature-sensitive (Ts6) peroxisome-deficient (per) mutant of Hansenula polymorpha. The subcellular location of PMP47 appeared to be dependent on the level of expression. At low expression levels PMP47 was sorted to the peroxisomal membrane; however, in Ts6 cells grown at restrictive temperatures (which lack intact peroxisomes) PMP47 was solely located in small cytosolic aggregates, together with homologous H. polymorpha PMP's. At enhanced expression levels, however, part of the protein also became incorporated into mitochondria, both in transformed WT and Ts6 cells.

Blotting, Western↗
PubMed1993

Synthesis and subcellular location of peroxisomal membrane proteins in a peroxisome-deficient mutant of the yeast Hansenula polymorpha.

We have studied the synthesis and subcellular location of peroxisomal membrane proteins (PMPs) in cells of a peroxisome-deficient (per) mutant of the methylotrophic yeast Hansenula polymorpha. Western blot analysis of methanol-induced cells of the per mutant, which had been growing in a continuous culture on a glucose/methanol mixture, indicated that various PMPs were normally synthesized. As in wild type (WT) cells, the levels of PMP synthesis appeared to be dependent on specific cultivation conditions, e.g. the carbon source used for growth. In contrast to WT controls, PMPs in methanol-induced per mutants were not subject to proteolytic degradation. Biochemical and immuno(cyto)chemical studies suggested that the PMPs in methanol-induced per cells were located in small proteinaceous aggregates, separated from peroxisomal matrix proteins that were also present in the cytosol. Vesicular membranous structures, resembling the morphology of intact peroxisomes, were never detected irrespective of the growth conditions employed.

Cell Fractionation↗
PubMed1993

Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles.

We have identified two temperature-sensitive peroxisome-deficient mutants of Hansenula polymorpha (ts6 and ts44) within a collection of ts mutants which are impaired for growth on methanol at 43 degrees C but grow well at 35 degrees C. In both strains peroxisomes were completely absent in cells grown at 43 degrees C; the major peroxisomal matrix enzymes alcohol oxidase, dihydroxyacetone synthase and catalase were synthesized normally but assembled into the active enzyme protein in the cytosol. As in wild-type cells, these enzymes were present in peroxisomes under permissive growth conditions (< or = 37 degrees C). However, at intermediate temperatures (38-42 degrees C) they were partly peroxisome-bound and partly resided in the cytosol. Genetic analysis revealed that both mutant phenotypes were due to monogenic recessive mutations mapped in the same gene, designated PER13. After a shift of per13-6ts cells from restrictive to permissive temperature, new peroxisomes were formed within 1 h. Initially one--or infrequently a few--small organelles developed which subsequently increased in size and multiplied by fission during prolonged permissive growth. Neither mature peroxisomal matrix nor membrane proteins, which were present in the cytosol prior to the temperature shift, were incorporated into the newly formed organelles. Instead, these proteins remained unaffected (and active) in the cytosol concomitant with further peroxisome development. Thus in H.polymorpha alternative mechanisms of peroxisome biogenesis may be possible in addition to multiplication by fission upon induction of the organelles by certain growth substrates.

Blotting, Western↗
PubMed1993

Peroxisomal amine oxidase of Hansenula polymorpha does not require its SRL-containing C-terminal sequence for targeting.

Amine oxidase (AMO) is a peroxisomal matrix protein of Hansenula polymorpha, which is induced during growth of the yeast in media containing primary amines as a sole nitrogen source. The deduced amino acid sequence of the protein contains an SRL sequence at nine amino acids from the C-terminus. In this study, we have examined the possible role of the SRL motif in sorting of AMO to peroxisomes by mutating the corresponding gene sequence. For this purpose, we have developed a DNA construct that is specifically integrated into the AMO locus of the H. polymorpha genome, placing the mutant gene under the control of the endogenous AMO promoter and eliminating expression of the wild-type gene. Analysis of a stable transformant, containing the desired gene configuration, showed that mutation of the C-terminal sequence neither interfered with correct targeting of the protein into the peroxisome nor displayed significant effects on its activity. From this, it was concluded that the SRL-containing C-terminus is not essential for peroxisomal targeting of AMO in H. polymorpha.

Amine Oxidase (Copper-Containing)↗
PubMed1993

The in vitro permeability of yeast peroxisomal membranes is caused by a 31 kDa integral membrane protein.

A major 31 kDa integral peroxisomal membrane protein (PMP31) of Hansenula polymorpha was purified to homogeneity from isolated peroxisomal membranes by FPLC after solubilization by Triton X-100. Biochemical analysis indicated that this protein, which showed cross-reactivity with antibodies against the 31 kDa porin of the mitochondrial outer membrane of Saccharomyces cerevisiae, had pore-forming properties. Firstly, proteoliposomes composed of asolectin and purified PMP31 showed selective permeability, determined as the [14C]sucrose/[3H]dextran leakage ratios. Furthermore, the generation of a delta psi by potassium diffusion gradients was negatively affected by the presence of PMP31 in asolectin liposomes. A similar effect was observed in proteoliposomes containing purified cytochrome c oxidase as a delta psi generating system. Control experiments confirmed that the observed leakage is significant and introduced by the incorporation of PMP31 protein. Selective sucrose leakage was abolished in samples pretreated with glutaraldehyde; an identical effect of glutaraldehyde was, however, not observed for the membrane potential measurements.

Cell Membrane Permeability↗
PubMed1993

Structural and functional aspects of peroxisomal membranes in yeasts.

The peroxisomal membrane compartmentalizes specific metabolic functions in the intermediary metabolism of various aerobic eukarya. In yeast, peroxisomal membranes are typified by their small width (+/- 7-8 nm) and absence of large integral membrane proteins in freeze-etch replicas. They show a unique polypeptide profile which, in contrast to their phospholipid composition, differs from that of other membranes in the cell. Part of these proteins are substrate-inducible and are probably related to specific peroxisomal function(s). In vivo, the observed proton motive force across the peroxisomal membrane may play a role in the function of the organelle in that it contributes to the driving force required for selective transport of various enzyme substrates and/or metabolic intermediates. To date only few peroxisomal membrane proteins (PMPs) have been functionally characterized. A major constitutive 31-kDa PMP present in the peroxisomal membrane of Hansenula polymorpha has been purified and was shown to display pore-forming properties. In addition, a peroxisomal H(+)-ATPase has been identified which most probably is involved in the generation/maintenance of the in vivo pH gradient across the peroxisomal membrane. Other functions of peroxisomal membrane proteins remain obscure although the first genes encoding yeast PMPs are now being cloned and sequenced. Studies on peroxisome-deficient yeast mutants revealed that specific peroxisome functions are strictly dependent on the intactness of the peroxisomal membrane. In this contribution several examples are presented of metabolic disorders due to peroxisomal malfunction in yeast.

Fungal Proteins↗
PubMed1993

Isolation of Typical Marine Bacteria by Dilution Culture: Growth, Maintenance, and Characteristics of Isolates under Laboratory Conditions.

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 x 10 to 1.07 x 10 cells per liter. The mean cell volume varied between 0.042 and 0.074 mum, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 x 10 times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 mum and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids (a degrees (A), 1,860 liters/g of cells per h), but not for glucose or alanine as the sole source of carbon and energy (a degrees (A), +/- 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.

Journal Article↗
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↗