Human catalase is imported and assembled in peroxisomes of <i>Saccharomyces cerevisiae</i>

Hoop, M. J. De; Holtman, W.; Ab, Geert

doi:10.1002/yea.320090108

Cited by 5 publications

(3 citation statements)

References 59 publications

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“…In contrast to mammalian cells which only express a single type of catalase in peroxisomes, yeast cells (S. cerevisiae) possess two distinct catalase isoenzymes, an atypical peroxisomal catalase A and a typical cytosolic catalase T, which both play an essential role in protecting yeast cells against oxidative stress [35]. However besides peroxisomes, mitochondria represent another compartment where most cellular ROS are being produced, and recently we presented evidence indicating that yeast mitochondria might indeed be the third compartment in which catalase activity can be found [9].…”

Section: Discussionmentioning

confidence: 99%

Dual targeting of yeast catalase A to peroxisomes and mitochondria

Petrova

Drescher

Kujumdzieva

et al. 2004

Biochemical Journal

122

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Yeast catalase A (Cta1p) contains two peroxisomal targeting signals (SSNSKF) localized at its C-terminus and within the N-terminal third of the protein, which both can target foreign proteins to peroxisomes. In the present study we demonstrated that Cta1p can also enter mitochondria, although the enzyme lacks a classical mitochondrial import sequence. Cta1p co-targeting was studied in a catalase A null mutant after growth on different carbon sources, and expression of a Cta1p-GFP (green fluorescent protein)-fusion protein or a Cta1p derivative containing either a c-Myc epitope (Cta1p(myc)) or a SKF-extended tag (Cta1p(myc-SKF)). Peroxisomal and mitochondrial co-import of catalase A were tested qualitatively by fluorescence microscopy and functional complementation of a Delta cta1 null mutation, and quantitatively by subcellular fractionation followed by Western blot analysis and enzyme activity assays. Efficient Cta1p import into peroxisomes was observed when cells were cultivated under peroxisome-inducing conditions (i.e. growth on oleate), whereas significant co-import of Cta1p-GFP into mitochondria occurred when cells were grown under respiratory conditions that favour oxygen stress and ROS (reactive oxygen species) accumulation within this organelle. In particular, when cells were grown on the non-fermentable carbon source raffinose, respiration is maximally enhanced, and catalase A was efficiently targeted to the mitochondrial matrix where it presumably functions as scavenger of H2O2 and mitochondrial-derived ROS.

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Section: Discussionmentioning

confidence: 99%

Dual targeting of yeast catalase A to peroxisomes and mitochondria

Petrova

Drescher

Kujumdzieva

et al. 2004

Biochemical Journal

122

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“…The 5,900-bp BamHI-SacI fragment of pY6, containing the entire MAL61 gene, was ligated into pBluescript SK Ϫ (Stratagene, La Jolla, Calif.). MAL61 was isolated from the resulting plasmid by digestion with HindIII and subsequently ligated into YEp13 (5), containing the ADC1 promoter and terminator located on a BamHI fragment and separated by a unique HindIII site (11).…”

Section: Methodsmentioning

confidence: 99%

Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes

et al. 1995

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For maltose uptake in Saccharomyces cerevisiae, multiple kinetic forms of transport as well as inhibition of transport by high concentrations of maltose at the trans side of the plasma membrane have been described. Most of these studies were hampered by a lack of genetically well-defined mutants and/or the lack of an artificial membrane system to study translocation catalysis in vitro. A genetically well-defined S. cerevisiae strain lacking the various MAL loci was constructed by gene disruption. Expression of the maltose transport protein (Mal61p) was studied by using various plasmid vectors that differed in copy number and/or type of promoter. The expression levels were quantitated by immunoblotting with antibodies generated against the N-terminal half of Mal61p. The levels of expression as well as the initial uptake rates were increased 20-fold compared with those in a yeast strain carrying only one chromosomal MAL locus. Similar results were obtained when the transport activities were compared in hybrid membranes of the corresponding strains. To generate a proton motive force, isolated membranes were fused with liposomes containing cytochrome c oxidase as a proton pump. Fusion was achieved by a cycle of freeze-thawing, after which the hybrid membranes were passed through a filter with a defined pore size to obtain unilamellar membrane vesicles. Proton motive force-driven maltose uptake, maltose efflux down the concentration gradient, and equilibrium exchange of maltose in the hybrid membranes vesicles have been analyzed. The data indicate that maltose transport by the maltose transporter is kinetically monophasic and fully reversible under all conditions tested.

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“…This prompted speculation that tripeptide PTSs might be able to function at internal locations in some instances (25). Also of interest is the observation that even though the COOH-terminal end of human catalase shows no significant sequence homology to that of yeast catalase A, the transgenically expressed human protein is targeted to yeast peroxisomes in vivo (14), implying evolutionary conservation of the peroxisomal sorting of this protein. In this paper, we address the molecular basis of this conservation of targeting by analyzing the sequences within human catalase responsible for targeting of this protein to yeast and human peroxisomes.…”

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confidence: 99%

Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal peroxisomal targeting sequence.

Purdue

Lazarow

1996

The Journal of Cell Biology

153

123

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Abstract. We have identified a novel peroxisomal targeting sequence (PTS) at the extreme COOH terminus of human catalase. The last four amino acids of this protein (-KANL) are necessary and sufficient to effect targeting to peroxisomes in both human fibroblasts and Saccharomyces cerevisiae, when appended to the COOH terminus of the reporter protein, chloramphenicol acetyl transferase. However, this PTS differs from the extensive family of COOH-terminal PTS tripeptides collectively termed PTS1 in two major aspects. First, the presence of the uncharged amino acid, asparagine, at the penultimate residue of the human catalase PTS is highly unusual, in that a basic residue at this position has been previously found to be a common and critical feature of PTS1 signals. Nonetheless, this asparagine residue appears to constitute an important component of the catalase PTS, in that replacement with aspartate abolished peroxisomal targeting (as did deletion of the COOH-terminal four residues). Second, the human catalase PTS comprises more than the COOH-terminal three amino acids, in that COOH-terminal -ANL cannot functionally replace the PTS1 signal -SKL in targeting a chloramphenicol acetyl transferase fusion protein to peroxisomes. The critical nature of the fourth residue from the COOH terminus of the catalase PTS (lysine) is emphasized by the fact that substitution of this residue with a variety of other amino acids abolished or reduced peroxisomal targeting. Targeting was not reduced when this lysine was replaced with arginine, suggesting that a basic amino acid at this position is required for maximal functional activity of this PTS. In spite of these unusual features, human catalase is sorted by the PTS1 pathway, both in yeast and human cells. Disruption of the PASIO gene encoding the S. cerevisiae PTS1 receptor resulted in a cytosolic location of chloramphenicol acetyl transferase appended with the human catalase PTS, as did expression of this protein in cells from a neonatal adrenoleukodystrophy patient specifically defective in PTS1 import. Furthermore, through the use of the two-hybrid system, it was demonstrated that both the PASIO gene product (Pasl0p) and the human PTS1 receptor can interact with the COOH-terminal region of human catalase, but that ithis interaction is abolished by substitutions at the penultimate residue (asparagine-toaspartate) and at the fourth residue from the COOH terminus (lysine-to-glycine) which abolish PTS functionality. We have found no evidence of additional targeting information elsewhere in the human catalase protein. An internal tripeptide (-SHL-, which conforms to the mammalian PTS1 consensus) located nine to eleven residues from the COOH terminus has been excluded as a functional PTS. Additionally, in contrast to the situation for S. cerevisiae catalase A, which contains an internal PTS in addition to a COOH-terminal PTS1, human catalase lacks such a redundant PTS, as evidenced by the exclusive cytosolic location of human catalase mutat+d in the COOH-terminal PTS. Consistent wi...

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Human catalase is imported and assembled in peroxisomes of Saccharomyces cerevisiae

Cited by 5 publications

References 59 publications

Dual targeting of yeast catalase A to peroxisomes and mitochondria

Dual targeting of yeast catalase A to peroxisomes and mitochondria

Overexpression of Mal61p in Saccharomyces cerevisiae and characterization of maltose transport in artificial membranes

Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal peroxisomal targeting sequence.

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