Protein Transport into Higher Plant Peroxisomes (In Vitro Import Assay Provides Evidence for Receptor Involvement)

Brickner, Donna Garvey; Harada, John J.; Olsen, Laura J.

doi:10.1104/pp.113.4.1213

Cited by 32 publications

(34 citation statements)

References 34 publications

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“…Organelles from etiolated pumpkin cotyledon extracts were separated by sucrose density centrifugation. To remove the contamination of cytosolic proteins that might be present in the solution or merely associated with the peroxisomal membrane, the isolated peroxisomes were treated with proteinase K. In this way, the peroxisomal proteins shielded from protease by the peroxisomal membrane were not digested (34,35). The identity of the peroxisomal fractions was proved by measuring the catalase activity (35).…”

Section: Resultsmentioning

confidence: 99%

Hydrogen peroxide homeostasis: Activation of plant catalase by calcium/calmodulin

Yang

Poovaiah

2002

Proc. Natl. Acad. Sci. U.S.A.

406

242

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Environmental stimuli such as UV, pathogen attack, and gravity can induce rapid changes in hydrogen peroxide (H2O2) levels, leading to a variety of physiological responses in plants. Catalase, which is involved in the degradation of H2O2 into water and oxygen, is the major H2O2-scavenging enzyme in all aerobic organisms. A close interaction exists between intracellular H2O2 and cytosolic calcium in response to biotic and abiotic stresses. Studies indicate that an increase in cytosolic calcium boosts the generation of H2O2. Here we report that calmodulin (CaM), a ubiquitous calcium-binding protein, binds to and activates some plant catalases in the presence of calcium, but calcium͞CaM does not have any effect on bacterial, fungal, bovine, or human catalase. These results document that calcium͞CaM can down-regulate H2O2 levels in plants by stimulating the catalytic activity of plant catalase. Furthermore, these results provide evidence indicating that calcium has dual functions in regulating H2O2 homeostasis, which in turn influences redox signaling in response to environmental signals in plants.

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Section: Resultsmentioning

confidence: 99%

Hydrogen peroxide homeostasis: Activation of plant catalase by calcium/calmodulin

Yang

Poovaiah

2002

Proc. Natl. Acad. Sci. U.S.A.

406

242

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“…However, the mechanism by which matrix proteins are translocated into the organelle has not yet been defined. In vitro experiments using isolated plant peroxisomes suggest the existence of a plant PTS1 receptor similar to the human and yeast Pex5p (27,28).…”

Section: Example the Motifs [A͞c͞p͞s]-[k͞ R]-[i͞l͞m] And [A͞c͞g͞s͞t]mentioning

confidence: 99%

Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5

Kragler

Lametschwandtner

Christmann

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

108

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Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (Nt-PEX5) based on the protein's interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

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“…Radiolabeled glycolate oxidase and AtSOX were synthesized in a cell-free rabbit reticulocyte lysate system (Promega) in the presence of [ 35 S]methionine. The efficiency of translation was assessed by trichloroacetic acid precipitation onto glass fiber filters followed by ethanol washes and liquid scintillation counting (29).…”

Section: Chemicals and Reagents-[n-methyl-mentioning

confidence: 99%

“…Isolation of Pumpkin Glyoxysomes-Glyoxysomes were isolated from pumpkin cotyledons as described previously (29). The glyoxysomal pellet was resuspended in isolation buffer (Hepes-KOH, pH 6.0, 0.3 M mannitol) to a final concentration of 35 mg/ml total protein.…”

Section: Chemicals and Reagents-[n-methyl-mentioning

confidence: 99%

“…Protease treatment was for 30 min on ice; reactions were stopped by adding EDTA (10 mM final concentration) to inhibit the thermolysin. The protease-treated glyoxysomes were then repurified on a sucrose cushion, solubilized in SDS sample buffer, and subjected to SDS-PAGE as described (29). Note that only proteins protected by an intact glyoxysomal membrane would be protease-resistant.…”

Section: Chemicals and Reagents-[n-methyl-mentioning

confidence: 99%

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Characterization and Metabolic Function of a Peroxisomal Sarcosine and Pipecolate Oxidase from Arabidopsis

Goyer

Johnson

Olsen

et al. 2004

Journal of Biological Chemistry

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Sarcosine oxidase (SOX) is known as a peroxisomal enzyme in mammals and as a sarcosine-inducible enzyme in soil bacteria. Its presence in plants was unsuspected until the Arabidopsis genome was found to encode a protein (AtSOX) with ϳ33% sequence identity to mammalian and bacterial SOXs. When overexpressed in Escherichia coli, AtSOX enhanced growth on sarcosine as sole nitrogen source, showing that it has SOX activity in vivo, and the recombinant protein catalyzed the oxidation of sarcosine to glycine, formaldehyde, and H 2 O 2 in vitro. AtSOX also attacked other N-methyl amino acids and, like mammalian SOXs, catalyzed the oxidation of L-pipecolate to ⌬ 1 -piperideine-6-carboxylate. Like bacterial monomeric SOXs, AtSOX was active as a monomer, contained FAD covalently bound to a cysteine residue near the C terminus, and was not stimulated by tetrahydrofolate. Although AtSOX lacks a typical peroxisome-targeting signal, in vitro assays established that it is imported into peroxisomes. Quantitation of mRNA showed that AtSOX is expressed at a low level throughout the plant and is not sarcosine-inducible. Consistent with a low level of AtSOX expression, Arabidopsis plantlets slowly metabolized supplied [ 14 C]sarcosine to glycine and serine. Gas chromatography-mass spectrometry analysis revealed low levels of pipecolate but almost no sarcosine in wild type Arabidopsis and showed that pipecolate but not sarcosine accumulated 6-fold when AtSOX expression was suppressed by RNA interference. Moreover, the pipecolate catabolite ␣-aminoadipate decreased 30-fold in RNA interference plants. These data indicate that pipecolate is the endogenous substrate for SOX in plants and that plants can utilize exogenous sarcosine opportunistically, sarcosine being a common soil metabolite.

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Protein Transport into Higher Plant Peroxisomes (In Vitro Import Assay Provides Evidence for Receptor Involvement)

Cited by 32 publications

References 34 publications

Hydrogen peroxide homeostasis: Activation of plant catalase by calcium/calmodulin

Hydrogen peroxide homeostasis: Activation of plant catalase by calcium/calmodulin

Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5

Characterization and Metabolic Function of a Peroxisomal Sarcosine and Pipecolate Oxidase from Arabidopsis

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