Rapid identification and characterization of peroxisomal assembly mutants in <i>Yarrowia lipolytica</i>

Nuttley, William M.; Brade, Anthony; Eitzen, Gary; Glover, John R.; Aitchison, John D.; Rachubinski, Richard A.; Gaillardin, Claude

doi:10.1002/yea.320090506

Cited by 78 publications

(85 citation statements)

References 43 publications

(33 reference statements)

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“…The PEX19 gene was isolated by functional complementation of the pex19-1 strain with a Y. lipolytica genomic DNA library in the autonomously replicating plasmid, pINA445 (Nuttley et al, 1993). Total DNA was isolated from colonies that recovered growth on YNO and was used to transform E. coli for plasmid recovery.…”

Section: Cloning Sequencing and Integrative Disruption Of The Pex19mentioning

confidence: 99%

“…The pex19-1 mutant strain was isolated from randomly mutagenized wild-type E122 cells as described previously (Nuttley et al, 1993). The PEX19 gene was isolated by functional complementation of the pex19-1 strain with a Y. lipolytica genomic DNA library in the autonomously replicating plasmid, pINA445 (Nuttley et al, 1993).…”

Section: Cloning Sequencing and Integrative Disruption Of The Pex19mentioning

confidence: 99%

“…Growth was at 30°C. Diploid strains were generated as described previously (Nuttley et al, 1993). Strains containing plasmids were grown on minimal medium (YND or YNO).…”

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Yarrowia lipolyticaCells Mutant for the Peroxisomal Peroxin Pex19p Contain Structures Resembling Wild-Type Peroxisomes

Lambkin

Rachubinski

2001

MBoC

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PEX genes encode peroxins, which are proteins required for peroxisome assembly. The PEX19 gene of the yeast Yarrowia lipolytica was isolated by functional complementation of the oleic acid-nonutilizing strain pex19-1 and encodes Pex19p, a protein of 324 amino acids (34,822 Da). Subcellular fractionation and immunofluorescence microscopy showed Pex19p to be localized primarily to peroxisomes. Pex19p is detected in cells grown in glucose-containing medium, and its levels are not increased by incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. pex19 cells preferentially mislocalize peroxisomal matrix proteins and the peripheral intraperoxisomal membrane peroxin Pex16p to the cytosol, although small amounts of these proteins could be reproducibly localized to a subcellular fraction enriched for peroxisomes. In contrast, the peroxisomal integral membrane protein Pex2p exhibits greatly reduced levels in pex19 cells compared with its levels in wild-type cells. Importantly, pex19 cells were shown by electron microscopy to contain structures that resemble wild-type peroxisomes in regards to size, shape, number, and electron density. Subcellular fractionation and isopycnic density gradient centrifugation confirmed the presence of vesicular structures in pex19 mutant strains that were similar in density to wild-type peroxisomes and that contained profiles of peroxisomal matrix and membrane proteins that are similar to, yet distinct from, those of wild-type peroxisomes. Because peroxisomal structures form in pex19 cells, Pex19p apparently does not function as a peroxisomal membrane protein receptor in Y. lipolytica. Our results are consistent with a role for Y. lipolytica Pex19p in stabilizing the peroxisomal membrane. INTRODUCTIONPeroxisomes, together with the glyoxysomes of plants and the glycosomes of trypanosomes, make up the microbody family of organelles (de Duve, 1996). In electron micrographs, peroxisomes appear spherical in shape, surrounded by a single unit membrane and containing a granular matrix and sometimes a paracrystalline core. The functions of peroxisomes vary depending on the organism, cell type, and physiological conditions. Functions that have been conserved from yeast to humans include the ␤-oxidation of fatty acids and the decomposition of hydrogen peroxide by catalase (for reviews, see Lazarow and Fujiki, 1985;van den Bosch et al., 1992). Proteins that are required for the proper functioning and biogenesis of peroxisomes have collectively been termed peroxins .The importance of peroxisomes for human growth and development is underscored by a group of genetic disorders known as the peroxisome biogenesis disorders (PBD), including Zellweger syndrome, rhizomelic chondrodysplasia punctata, and neonatal adrenoleukodystrophy, in which peroxisomes fail to assemble normally (for reviews, see Lazarow and Moser, 1994;Fujiki, 1997Fujiki, , 2000. A great deal of attention has been paid recently to defining the molecular bases of these diseases, in particular...

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Section: Cloning Sequencing and Integrative Disruption Of The Pex19mentioning

confidence: 99%

Section: Cloning Sequencing and Integrative Disruption Of The Pex19mentioning

confidence: 99%

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Yarrowia lipolyticaCells Mutant for the Peroxisomal Peroxin Pex19p Contain Structures Resembling Wild-Type Peroxisomes

Lambkin

Rachubinski

2001

MBoC

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“…The pex23-1 mutant strain was isolated from randomly mutagenized Y. lipolytica strain E122 as described previously (Nuttley et al, 1993). The PEX23 gene was isolated by functional complementation of the pex23-1 strain with a Y. lipolytica genomic DNA library in the autonomously replicating E. coli shuttle vector pINA445 (Nuttley et al, 1993).…”

Section: Cloning Sequencing and Integrative Disruption Of The Pex23mentioning

confidence: 99%

“…The PEX23 gene was isolated by functional complementation of the pex23-1 strain with a Y. lipolytica genomic DNA library in the autonomously replicating E. coli shuttle vector pINA445 (Nuttley et al, 1993). Leu ϩ transformants were replica plated onto selective YNO agar plates and screened for their ability to use oleic acid as a sole carbon source.…”

Section: Cloning Sequencing and Integrative Disruption Of The Pex23mentioning

confidence: 99%

Mutants of theYarrowia lipolytica PEX23Gene Encoding an Integral Peroxisomal Membrane Peroxin Mislocalize Matrix Proteins and Accumulate Vesicles Containing Peroxisomal Matrix and Membrane Proteins

Brown

Titorenko

Rachubinski

2000

MBoC

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pex mutants are defective in peroxisome assembly. The mutant strain pex23-1 of the yeast Yarrowia lipolytica lacks morphologically recognizable peroxisomes and mislocalizes all peroxisomal matrix proteins investigated preferentially to the cytosol. pex23 strains accumulate vesicular structures containing both peroxisomal matrix and membrane proteins. The PEX23 gene was isolated by functional complementation of the pex23-1 strain and encodes a protein, Pex23p, of 418 amino acids (47,588 Da). Pex23p exhibits high sequence similarity to two hypothetical proteins of the yeastSaccharomyces cerevisiae. Pex23p is an integral membrane protein of peroxisomes that is completely, or nearly completely, sequestered from the cytosol. Pex23p is detected at low levels in cells grown in medium containing glucose, and its levels are significantly increased by growth in medium containing oleic acid, the metabolism of which requires intact peroxisomes.

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Cloning and characterization of the peroxisomal acyl CoA oxidaseACO3 gene from the alkane-utilizing yeastYarrowia lipolytica

Wang

Clainche

Dall

et al. 1998

Yeast

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The ACO3 gene, which encodes one of the acyl‐CoA oxidase isoenzymes, was isolated from the alkane‐utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701‐amino acid protein very similar to other ACOs, 67·5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl‐CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild‐type strain, suggested that ACO3 encodes a short chain acyl‐CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli. © 1998 John Wiley & Sons, Ltd.

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Rapid identification and characterization of peroxisomal assembly mutants in Yarrowia lipolytica

Cited by 78 publications

References 43 publications

Yarrowia lipolyticaCells Mutant for the Peroxisomal Peroxin Pex19p Contain Structures Resembling Wild-Type Peroxisomes

Yarrowia lipolyticaCells Mutant for the Peroxisomal Peroxin Pex19p Contain Structures Resembling Wild-Type Peroxisomes

Mutants of theYarrowia lipolytica PEX23Gene Encoding an Integral Peroxisomal Membrane Peroxin Mislocalize Matrix Proteins and Accumulate Vesicles Containing Peroxisomal Matrix and Membrane Proteins

Cloning and characterization of the peroxisomal acyl CoA oxidaseACO3 gene from the alkane-utilizing yeastYarrowia lipolytica

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