Targeting and Subcellular Localization of Toxoplasma gondii Catalase
IDENTIFICATION OF PEROXISOMES IN AN APICOMPLEXAN PARASITE*
- From the Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8022
Abstract
We sought to identify and characterize peroxisomes in the apicomplexan parasite Toxoplasma gondii. To initiate this process, we first cloned and sequenced the gene forT. gondii catalase (EC 1.11.1.6), a marker enzyme for peroxisomes in eukaryotic cells. The gene predicts a protein of 57.2 kDa and 502 amino acids and has a strong homology to other eukaryotic catalases. A polyclonal antiserum raised against a glutathioneS-transferase fusion protein recognized a single band with a molecular mass of 63 kDa by immunoblot. By immunofluorescenceT. gondii catalase is present primarily in a punctate staining pattern anterior to the parasite nucleus. This compartment is distinguishable from other parasite organelles, namely micronemes, rhoptries, dense granules, and the apicoplast. Cytochemical visualization of catalase using diaminobenzidine precipitation gives a vesicular staining pattern anterior to the nucleus at the light level and round, vesicular structures with an estimated diameter of 100–300 nm by electron microscopy. T. gondii catalase has a putative C-terminal peroxisomal targeting signal in the last 3 amino acids (-AKM). Expression of T. gondii catalase in mammalian cells results in peroxisomal localization, whereas a construct lacking the targeting signal remains in the cytosol. Furthermore, addition of -AKM to the C terminus of chloramphenicol acetyltransferase is sufficient to target this protein to peroxisomes. These results provide the first evidence for peroxisomes in Apicomplexan parasites.
Footnotes
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↵* This work was supported by National Institutes of Health Grant A130060 and a Burroughs Wellcome Scholar award in Molecular Parasitology (to K. A. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) and .
The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI accession numbers 179950, 1078837, and 115709.
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↵‡ To whom correspondence should be addressed: Infectious Diseases Section, Dept. of Internal Medicine, 808 LCI, 333 Cedar St., New Haven, CT 06520-8022. Tel.: 203-785-4140; Fax: 203-785-3864; E-mail: keith.joiner@yale.edu.
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↵2 I. Coppens, A. Sinai, D. Voelker, and K. A. Joiner, unpublished observations.
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↵3 E. Mui, B. Samuel, D. Mack, C. Roberts, C. Pope, F. Roberts, D. Trelease, W. Milhous, D. Kyle, S. Tzipori, and R. McLeod, Fifth Toxoplasma Conference, Marshall, CA, May 1–6, 1999.
- Abbreviations:
- PTS
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peroxisomal targeting signal
- PBS
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phosphate buffered saline
- DAB
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diaminobenzidine
- GST
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glutathione S-transferase, T/S, Teorell-Stenhagen buffer
- CAT
-
chloramphenicol acetyltransferase
- IFA
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immunofluorescence assay
- RT-PCR
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reverse transcriptase-polymerase chain reaction
- CHO
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Chinese hamster ovary
- PIPES
-
1,4-piperazinediethanesulfonic acid
- aa
-
amino acids
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- Received September 2, 1999.
- Revision received September 29, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
