scholarly journals Peroxisomes induced in Candida boidinii by methanol, oleic acid and D-alanine vary in metabolic function but share common integral membrane proteins

1990 ◽  
Vol 97 (1) ◽  
pp. 193-204 ◽  
Author(s):  
J.M. Goodman ◽  
S.B. Trapp ◽  
H. Hwang ◽  
M. Veenhuis
Keyword(s):  
Oleic Acid ◽  
Membrane Proteins ◽  
Carbon Sources ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Acid Oxidase ◽  
General Function ◽  
Amino Acid Oxidase ◽  
Peroxisomal Membrane ◽  
Methanol Metabolism

Peroxisomes massively proliferate in the methylotrophic yeast Candida boidinii when cultured on methanol as the only carbon and energy source. These organelles contain enzymes that catalyze the initial reactions of methanol utilization. The membranes contain abundant proteins of unknown function; their apparent molecular masses are 20, 31, 32 and 47 × 10(3) Mr and are termed PMP20, PMPs31-32 and PMP47. Recently, we reported that peroxisomes in this yeast are also induced by oleic acid and D-alanine as carbon sources, and that these peroxisomes contain increased concentrations of the enzymes of fatty acid beta-oxidation or D-amino acid oxidase, respectively. This report extends these findings and further compares the enzyme composition from peroxisomes induced by methanol, oleic acid and D-alanine. the patterns of matrix proteins represented on SDS-polyacrylamide gels from peroxisomes induced by oleic acid or D-alanine were found to be very different from those of peroxisomes induced by methanol. In order to differentiate between membrane proteins that have specific functions in pathways of substrate utilization from those with more generalized functions, peroxisomal membranes from cultures grown on methanol, oleic acid or D-alanine were purified. Analysis of these fractions demonstrated that while PMP20 is found only in peroxisomes induced by methanol, the PMPs31-32 and PMP47 were the abundant peroxisomal membrane proteins (PMP) regardless of inducing substrate. The data strongly suggest that the function of PMP20 is related to methanol metabolism. In contrast, the functions of PMPs31-32 and PMP47 are ‘substrate-nonspecific’. We speculate that they may relate to the structure, assembly or general function of the organelle.

scholarly journals Peroxisomal Catalase in the Methylotrophic Yeast Candida boidinii: Transport Efficiency and Metabolic Significance

2001 ◽  
Vol 183 (21) ◽  
pp. 6372-6383 ◽  
Author(s):  
Hirofumi Horiguchi ◽  
Hiroya Yurimoto ◽  
Toh-Kheng Goh ◽  
Tomoyuki Nakagawa ◽  
Nobuo Kato ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
System Analysis ◽  
Fluorescent Protein ◽  
Carbon Sources ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Transport Efficiency ◽  
Green Fluorescent

ABSTRACT In this study we cloned CTA1, the gene encoding peroxisomal catalase, from the methylotrophic yeast Candida boidinii and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins. A strain from which CTA1 was deleted (cta1Δ strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and d-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism. Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus. Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and d-alanine-grown cells. In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 andd-amino acid oxidase, was found to localize exclusively in peroxisomes. A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence. Furthermore, GFP-Cta1pΔnkf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif. Since the main physiological function of catalase is degradation of H2O2, suboptimal efficiency of catalase import may confer an evolutionary advantage. We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.

Characterization and High-level Production of D-Amino Acid Oxidase in Candida boidinii

2001 ◽  
Vol 65 (3) ◽  
pp. 627-633 ◽  
Author(s):  
Hiroya YURIMOTO ◽  
Tetsuya HASEGAWA ◽  
Yasuyoshi SAKAI ◽  
Nobuo KATO
Keyword(s):  
Amino Acid ◽  
Candida Boidinii ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
High Level ◽  
Level Production

scholarly journals Peroxisomal assembly: membrane proliferation precedes the induction of the abundant matrix proteins in the methylotrophic yeast Candida boidinii

1990 ◽  
Vol 96 (4) ◽  
pp. 583-590
Author(s):  
M. Veenhuis ◽  
J.M. Goodman
Keyword(s):  
Early Stage ◽  
Morphological Changes ◽  
Polyclonal Antibodies ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Peroxisomal Membrane ◽  
Cell Induction ◽  
The Matrix ◽  
Dihydroxyacetone Synthase

Peroxisomes are massively induced when methylotrophic yeasts are cultured in medium containing methanol. These organelles contain enzymes that catalyze the initial steps of methanol assimilation. In Candida boidinii, a methylotrophic yeast, the peroxisomal matrix (internal compartment) is composed almost exclusively of two proteins, alcohol oxidase and dihydroxyacetone synthase; catalase is present in much lower abundance. Monoclonal and polyclonal antibodies are available against peroxisomal matrix and membrane proteins. These were utilized to correlate the induction of specific proteins with the morphological changes occurring during peroxisomal proliferation. Cells cultured in glucose-containing medium contain two to five small microbodies, which are identifiable by catalase staining and immunoreactivity with a monoclonal antibody against PMP47, an integral peroxisomal membrane protein. Three stages of proliferation can be distinguished when cells are switched to methanol as the carbon source. (1) There is an early stage (within 1 h) in which several peroxisomes develop from a preexisting organelle. This is accompanied by an increase in catalase activity and an induction of PMP47, but no detectable induction of alcohol oxidase or dihydroxyacetone synthase is observed. (2) From 1 to 2.5 h there is further division of these microbodies until up to 30 small peroxisomes generally are present in each of one or two clusters per cell. Induction of alcohol oxidase, dihydroxyacetone synthase and PMP20, a protein that is distributed in the matrix and membrane, is detectable during this time. Serial sections reveal that some peroxisomes remain uninduced while others undergo proliferation. Such sections also show no obvious connections between peroxisomes within clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Enhancement of L-amino Acid Oxidase Production by Bacillus Subtilis H-8 With Oxygen-vector and Asymmetric Degradation of DL Arginine to D-arginine

2020 ◽  
Author(s):  
peng xu ◽  
Changpei Pan ◽  
Gongcheng Cui ◽  
ChunYan Wei ◽  
Lijuan Wang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Oleic Acid ◽  
Amino Acid ◽  
Fermentation Broth ◽  
Cation Exchange Resin ◽  
Optical Purity ◽  
Phenyl Isothiocyanate ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Oxygen Vector

Abstract The Bacillus subtilis H-8 independently screened by our laboratory can produce L-amino acid oxidase (L-AAO), and DL-arginine can be degraded asymmetrically by suspending the wet bacteria in the degradation liquid. By adding oxygen-vectors to the fermentation medium, the collected amount of wet bacteria can be increased. Taking n-dodecane, n-hexadecane, oleic acid, paraffin, and n-hexane as oxygen-vectors, the optimal oxygen-vector oleic acid was 1.2% (v/v). The weight of wet cells increased by 66.83% compared with before, and the activity of L-AAO in fermentation broth increased by 38.88% compared with before. The standard sample DL-arginine was derivatized by phenyl isothiocyanate, and then subjected to high performance liquid chromatography(HPLC), and the obtained peak area and arginine content were used as standard curves to measure the DL-arginine. The content of D-arginine and L-arginine in the initial degradation solution was 50% each, and the bacterial cells are added to the initial degradation solution of DL-arginine. After 21 hours of reaction, L-arginine was completely Degraded, remaining 47% of D-arginine.D-alanine was easily extracted from the reaction solution using cation-exchange resin,after centrifugation, decolorization, concentration and vacuum drying, and the chemical and optical purity of the extracted d-alanine was 92.68 and 97.46%, respectively.

scholarly journals Pmp27 promotes peroxisomal proliferation.

1995 ◽  
Vol 129 (2) ◽  
pp. 345-355 ◽  
Author(s):  
P A Marshall ◽  
Y I Krimkevich ◽  
R H Lark ◽  
J M Dyer ◽  
M Veenhuis ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Sequence Similarity ◽  
Carbon Sources ◽  
Weight Of Evidence ◽  
Candida Boidinii ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Beta Oxidation ◽  
Peroxisomal Membrane ◽  
A Cell

Peroxisomes perform many essential functions in eukaryotic cells. The weight of evidence indicates that these organelles divide by budding from preexisting peroxisomes. This process is not understood at the molecular level. Peroxisomal proliferation can be induced in Saccharomyces cerevisiae by oleate. This growth substrate is metabolized by peroxisomal enzymes. We have identified a protein, Pmp27, that promotes peroxisomal proliferation. This protein, previously termed Pmp24, was purified from peroxisomal membranes, and the corresponding gene, PMP27, was isolated and sequenced. Pmp27 shares sequence similarity with the Pmp30 family in Candida boidinii. Pmp27 is a hydrophobic peroxisomal membrane protein but it can be extracted by high pH, suggesting that it does not fully span the bilayer. Its expression is regulated by oleate. The function of Pmp27 was probed by observing the phenotype of strains in which the protein was eliminated by gene disruption or overproduced by expression from a multicopy plasmid. The strain containing the disruption (3B) was able to grow on all carbon sources tested, including oleate, although growth on oleate, glycerol, and acetate was slower than wild type. Strain 3B contained peroxisomes with all of the enzymes of beta-oxidation. However, in addition to the presence of a few modestly sized peroxisomes seen in a typical thin section of a cell growing on oleate-containing medium, cells of strain 3B also contained one or two very large peroxisomes. In contrast, cells in a strain in which Pmp27 was overexpressed contained an increased number of normal-sized peroxisomes. We suggest that Pmp27 promotes peroxisomal proliferation by participating in peroxisomal elongation or fission.

scholarly journals Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

2015 ◽  
Vol 14 (3) ◽  
pp. 278-285 ◽  
Author(s):  
Saori Oda ◽  
Hiroya Yurimoto ◽  
Nobuhisa Nitta ◽  
Yu Sasano ◽  
Yasuyoshi Sakai
Keyword(s):  
Gene Expression ◽  
Carbon Sources ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Promoter Regions ◽  
Inducible Gene Expression ◽  
Content Type ◽  
Inducible Promoters ◽  
Hap Complex ◽  
Inducible Gene

ABSTRACT We identified genes encoding components of the Hap complex, CbHAP2 , CbHAP3 , and CbHAP5 , as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii . We found that the Cbhap2 Δ, Cbhap3 Δ, and Cbhap5 Δ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae , the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii . Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts.

scholarly journals Mutants of the Yeast Yarrowia lipolyticaDefective in Protein Exit from the Endoplasmic Reticulum Are Also Defective in Peroxisome Biogenesis

1998 ◽  
Vol 18 (5) ◽  
pp. 2789-2803 ◽  
Author(s):  
Vladimir I. Titorenko ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Oleic Acid ◽  
Membrane Proteins ◽  
Matrix Proteins ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Mutant Cells

ABSTRACT Mutations in the SEC238 and SRP54 genes of the yeast Yarrowia lipolytica not only cause temperature-sensitive defects in the exit of the precursor form of alkaline extracellular protease and of other secretory proteins from the endoplasmic reticulum and in protein secretion but also lead to temperature-sensitive growth in oleic acid-containing medium, the metabolism of which requires the assembly of functionally intact peroxisomes. The sec238A andsrp54KO mutations at the restrictive temperature significantly reduce the size and number of peroxisomes, affect the import of peroxisomal matrix and membrane proteins into the organelle, and significantly delay, but do not prevent, the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX1 and PEX6 genes, which encode members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like ATPases, not only affect the exit of precursor forms of secretory proteins from the endoplasmic reticulum but also prevent the exit of the peroxisomal membrane proteins Pex2p and Pex16p from the endoplasmic reticulum and cause the accumulation of an extensive network of endoplasmic reticulum membranes. None of the peroxisomal matrix proteins tested associated with the endoplasmic reticulum in sec238A,srp54KO, pex1-1, and pex6KO mutant cells. Our data provide evidence that the endoplasmic reticulum is required for peroxisome biogenesis and suggest that inY. lipolytica, the trafficking of some membrane proteins, but not matrix proteins, to the peroxisome occurs via the endoplasmic reticulum, results in their glycosylation within the lumen of the endoplasmic reticulum, does not involve transport through the Golgi, and requires the products encoded by the SEC238, SRP54,PEX1, and PEX6 genes.

scholarly journals A Methylotrophic Pathway Participates in Pectin Utilization by Candida boidinii

2000 ◽  
Vol 66 (10) ◽  
pp. 4253-4257 ◽  
Author(s):  
Tomoyuki Nakagawa ◽  
Tatsuro Miyaji ◽  
Hiroya Yurimoto ◽  
Yasuyoshi Sakai ◽  
Nobuo Kato ◽  
...  
Keyword(s):  
Methyl Ester ◽  
Alcohol Oxidase ◽  
Pectate Lyase ◽  
Methylotrophic Yeast ◽  
Pectin Methylesterase ◽  
Candida Boidinii ◽  
Methanol Metabolism ◽  
Genes Encoding ◽  
Dihydroxyacetone Synthase ◽  
In Cells

ABSTRACT The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.

scholarly journals Enhancement of L-amino Acid Oxidase Production by Bacillus Subtilis HLZ-68 With Oxygen-vector and Asymmetric Degradation of DL Arginine to D-arginine

2020 ◽  
Author(s):  
peng xu ◽  
Changpei Pan ◽  
Gongcheng Cui ◽  
ChunYan Wei ◽  
Lijuan Wang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Oleic Acid ◽  
Amino Acid ◽  
Fermentation Broth ◽  
Cation Exchange Resin ◽  
Optical Purity ◽  
Phenyl Isothiocyanate ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Oxygen Vector

Abstract The Bacillus subtilis HLZ-68 independently screened by our laboratory can produce L-amino acid oxidase (L-AAO), and DL-arginine can be degraded asymmetrically by suspending the wet bacteria in the degradation liquid. By adding oxygen-vectors to the fermentation medium, the collected amount of wet bacteria can be increased. Taking n-dodecane, n-hexadecane, oleic acid, paraffin, and n-hexane as oxygen-vectors, the optimal oxygen-vector oleic acid was 1.2% (v/v). The weight of wet cells increased by 66.83% compared with before, and the activity of L-AAO in fermentation broth increased by 38.88% compared with before. The standard sample DL-arginine was derivatized by phenyl isothiocyanate, and then subjected to high performance liquid chromatography(HPLC), and the obtained peak area and arginine content were used as standard curves to measure the DL-arginine. The content of D-arginine and L-arginine in the initial degradation solution was 50% each, and the bacterial cells are added to the initial degradation solution of DL-arginine. After 21 hours of reaction, L-arginine was completely Degraded, remaining 47% of D-arginine.D-alanine was easily extracted from the reaction solution using cation-exchange resin,after centrifugation, decolorization, concentration and vacuum drying, and the chemical and optical purity of the extracted d-alanine was 92.68 and 97.46%, respectively.

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