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  • 1
    ISSN: 0749-503X
    Keywords: yeast ; peroxisome biogenesis ; peroxisome-deficient mutant ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Expression of the peroxisome-deficient (Per-) phenotype by per mutants of Hansenula polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature-sensitive (ts) mutants, per13-6ts and per14-11ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild-type cells, namely D-alanine (for both mutants) or methylamine (for per14-11ts). These organelles displayed normal wild-type properties with respect to morphology, mode of development and protein composition.However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno) cytochemical analyses indicated that during growth of cells on methanol in the presence of either D-alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol.
    Additional Material: 4 Ill.
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  • 2
    ISSN: 0749-503X
    Keywords: peroxisome biogenesis ; peroxisomal protein import ; peroxisomal membrane protein ; PEX gene regulation ; Hansenula polymorpha ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: PEX3 encodes a 52 kDa peroxisomal membrane protein (PMP), essential for peroxisome biogenesis in the yeast Hansenula polymorpha. The relation between Pex3p levels and peroxisome formation was studied in wild type (WT) and Δpex3 strains expressing additional copies of PEX3 under control of a substrate-inducible promoter, namely the strong alcohol oxidase (PAOX) or the weaker amine oxidase (PAMO) promoter. In glucose-grown Δpex3 cells, containing PAOX. PEX3, Pex3p was undetectable and peroxisomes were absent. After induction of these cells on methanol, peroxisomes were rapidly formed. At Pex3p levels up to 7-10 times the values observed in WT controls normal peroxisomes were present. However, at further enhanced Pex3p levels a general matrix protein import defect was observed. This phenomenon was paralleled by aberrant peroxisome assembly and the formation of numerous small vesicles. These vesicles contained Pex3p, together with other H. polymorpha PMPs, but lacked the major matrix proteins which has accumulated in the cytosol. The implications of our results on PEX3 gene regulation and functioning of the peroxisomal matrix protein import machinery in H. polymorpha are discussed. © 1997 John Wiley & Sons, Ltd.
    Additional Material: 7 Ill.
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  • 3
    ISSN: 1432-0983
    Keywords: Key wordsPichia pastoris ; Peroxisome degradation ; Autophagy ; VPS15
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Methylotrophic yeasts contain large peroxisomes during growth on methanol. Upon exposure to excess glucose or ethanol these organelles are selectively degraded by autophagy. Here we describe the cloning of a Pichia pastoris gene (PpVPS15) involved in peroxisome degradation, which is homologous to Saccharomyces cerevisiae VPS15. In methanol-grown cells of a P. pastoris VPS15 deletion strain, the levels of peroxisomal marker enzymes remained high after addition of excess glucose or ethanol. Electron microscopic studies revealed that the organelles were not taken up by vacuoles, suggesting that PpVPS15 is required at an early stage in peroxisome degradation.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Microscopy Research and Technique 27 (1994), S. 284-293 
    ISSN: 1059-910X
    Keywords: Mitochondrion ; Contact sites ; Protein translocation ; Ribosomes ; Import intermediates ; Receptor proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Mitochondrial protein targeting includes both intramitochondrial sorting of proteins encoded by the organellar genome and import and subsequent sorting of nuclear encoded precursor proteins. Only a few proteins are encoded by the mitochondrial genome and synthesized in the organellar matrix. These include predominantly inner membrane proteins that are perhaps co-translationally inserted into this membrane. Biochemical data suggest that insertion into the inner membrane may be confined to the inner boundary membrane. Ultrastructurally, however, a preferential association of ribosomes with either inner boundary or cristae membranes has not been established.The majority of the mitochondrial proteins are nuclear encoded and synthesized as precursors in the cytosol. Electron microscopic studies revealed that import of precursor proteins is generally confined to sites where both mitochondrial envelope membranes are closely apposed. In line with these observations, biochemical studies indicated that precursor proteins destined for the inner membrane or matrix have to interact with the energized inner membrane to allow complete passage of the precursor through the outer membrane. As a consequence, the mitochondrial envelope membranes have to be in close proximity at protein import sites.In isolated mitochondria distinct sites (designated as contact sites) exist where both envelope membranes are closely apposed and presumably stably associated. In situ, however, mitochondrial boundary membranes are in close proximity over large areas that cover almost the entire mitochondrial periphery. Consequently, the relative area of the mitochondrial surface, where both boundary membranes are in sufficient proximity for allowing protein translocation, is generally larger in situ compared to that in isolated organelles.Immunocytochemical localization studies showed a rather random distribution of components of the mitochondrial protein translocation machinery over the entire mitochondrial surface and not confined to contact sites.Based on these ultrastructral data and recent biochemical findings we propose that mitochondrial protein import sites are dynamic in nature and include relatively labile regions of close association of the boundary membranes. In vitro, however, mitochondrial protein import may preferentially take place at or near the presumably stable contact sites. © 1994 Wiley-Liss, Inc.
    Additional Material: 3 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 6 (1990), S. 87-97 
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; methylotrophic yeast ; microbodies ; peroxisome-deficient mutants ; alcohol oxidase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: As a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 260 methanol-utilization-defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase.Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol- and ethanol utilizing-deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome-deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol.The mutations complemented and therefore were recessive mutations in different genes.
    Additional Material: 6 Ill.
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  • 6
    ISSN: 0749-503X
    Keywords: Hansenula polymorpha ; alcohol oxidase ; amine oxidase ; choline ; peroxisome-deficient mutant ; enzyme assembly ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have studied the expression of alcohol oxidase (AO) in a peroxisome-deficient mutant strain of Hansenula polymorpha. High levels of octameric, active AO (up to 3·0 U/mg protein) were detected in cells grown at low dilution rates in a glucose-limited chemostat in the presence of choline as the sole nitrogen source. Monomeric or other intermediate forms of AO were not detected in the mutant strain. This indicated that assembly of the protein into active octameric molecules in the cytosol was as efficient as in wild-type cells where this process is confined to the peroxisomal matrix. At relatively low rates of expression (less than 1 U/mg protein) AO was localized throughout the cytosol and, surprisingly, was also present inside the nucleus. However, at enhanced levels large crystalloids were formed. Generally one crystalloid was observed per cell, whereas smaller ones were occasionally found in developing buds. Also large crystalloids have been observed inside the nucleus. These crystalloids were not surrounded by a membrane. Based on the morphology of the molecules that constituted these crystalloids and the results of (immuno)cytochemical experiments we conclude that the crystalloids are composed of octameric AO molecules, arranged in a regular lattice, identical to the 3-dimensional architecture previously described for the crystalline matrix of peroxisomes in methanol-grown wild type cells of H. polymorpha. Attempts to purify the crystalloids by conventional fractionation methods failed, due to their apparent fragility; however, (immuno)cytochemical experiments revealed that catalase and dihydroxyacetone synthase were also associated with these structures.
    Additional Material: 15 Ill.
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  • 7
    ISSN: 0749-503X
    Keywords: heat shock proteins ; molecular chaperones ; stress tolerance ; peroxisome biogenesis ; Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was constitutively present. Peptides derived from both Hsp72 and Hsc74 showed sequence homology to the cytosolic Saccharomyces cerevisiae Hsp70s, Ssa1p and Ssa2p. The gene encoding Hsp72 (designated HSA1) was cloned, sequenced and used to construct HSA1 disruption and HSA1 overexpression strains. Comparison of the stress tolerances of these strains with those of wild-type H. polymorpha revealed that HSA1 overexpression negatively affected the tolerance of the cells to killing effects of temperature or ethanol, but enhanced the tolerance to copper and cadmium. The tolerance for other chemicals (arsenite, arsenate, H2O2) or to high osmolarity was unaffected by either deletion or overexpression of HSA1. The nucleotide sequence of HSA1 was submitted to the EMBL data library and given the Accession Number Z29379.
    Additional Material: 4 Ill.
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  • 8
    ISSN: 0749-503X
    Keywords: Yeast ; Hansenula polymorpha ; microbodies ; biogenesis ; PER genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroximal matrix proteins. These mutants are characterized by the presence of small intact peroxisomes, while the bulk of the peroxisomal matrix protein is not imported and resides in the cytosol (Pim- phenotype). Genetic analysis of back-crossed mutants revealed five different complementation groups, which were designated PERI-PER5. Mapping studies to determine the linkage relationships indicated that the observed Pim- phenotypes were determined by single recessive nuclear mutations.The different mutants had comparable phenotypes: (i) they were impaired to utilize methanol as the sole source of carbon and energy but grew well on various other compounds, including nitrogen sources, the metabolism of which is known to be mediated by peroxisome-borne enzymes in wild-type cells; (ii) all peroxisomal enzymes tested were induced, assembled and activated as in wild-type cells although their activities varied between the different representative mutants; (iii) all peroxisomal proteins, whether constitutive or inducible, were found both in the cytosol and in the small peroxisomes. These results suggest that a general, major import mechanism is affected in all mutants.
    Additional Material: 2 Ill.
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  • 9
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Additional Material: 11 Ill.
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  • 10
    ISSN: 0749-503X
    Keywords: peroxisome biogenesis ; peroxisomal protein import ; peroxisomal membrane protein ; peroxisome proliferation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Pex3p has been implicated in the biosynthesis of the peroxisomal membrane of the yeast Hansenula polymorpha. Here we show that in the initial stages of a sharp increase in Pex3p levels, induced in batch cultures of cells of a constructed H. polymorpha strain, which contained seven copies of PEX3 under control of the alcohol oxidase promoter (WT::PAOX.PEX37x), strongly interfered with normal peroxisome proliferation. Ultrastructural studies demonstrated that in such cells numerous small peroxisomes had developed, which were absent in wild-type controls. These organelles, which contained typical peroxisomal matrix and membrane proteins (alcohol oxidase, catalase, Pex3p, Pex10p and Pex14p), showed a relatively low density (1·18 g cm-3) after sucrose gradient centrifugation of WT::PAOX.PEX37x homogenates, compared to normal peroxisomes (1·23 g cm-3). We furthermore demonstrated that these early induced, small peroxisomes were protected against glucose-induced proteolytic degradation and did not fuse to form larger organelles. Remarkably, the induction of these small peroxisomes was paralleled by a partial defect in matrix protein import, reflected by the mislocalization of minor amounts of alcohol oxidase protein in the cytosol. However, when the cells were subsequently placed under conditions in which the synthesis of a new matrix enzyme (amine oxidase) was induced while simultaneously the excessive proliferation was repressed (by repression of the PAOX), amine oxidase protein was selectively incorporated into these organelles. This indicated that the small peroxisomes had regained a normal protein import capacity. Based on these results we argue that peroxisome proliferation and matrix protein import are coupled processes in H. polymorpha. © 1997 John Wiley & Sons, Ltd.
    Additional Material: 11 Ill.
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