Targeting and Processing of Nuclear-encoded Apicoplast Proteins in Plastid Segregation Mutants of Toxoplasma gondii

He, Cynthia Y.; Striepen, Boris; Pletcher, Charles H.; Murray, John M.; Roos, David S.

doi:10.1074/jbc.m102000200

Cited by 52 publications

(44 citation statements)

References 43 publications

(62 reference statements)

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“…PfALAD signal is sparse, although its presence in the apicoplast can be clearly seen Fig. 10, C and D. The distinctive four-membrane architecture that characterizes the apicoplast is reported to be fragile (35). This is the first report on the actual detection of an enzyme of the heme-biosynthetic pathway in the apicoplast.…”

Section: Localization Of Alad In the Parasitementioning

confidence: 86%

δ-Aminolevulinic Acid Dehydratase from Plasmodium falciparum

Dhanasekaran

Chandra

Sagar

et al. 2004

Journal of Biological Chemistry

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The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host ␦-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory. In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized. The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit M r of 46,000. The enzyme has an alkaline pH optimum of 8.0 to 9.0. rPfALAD does not require any metal ion for activity, although it is stimulated by 20 -30% upon addition of Mg 2؉ . The enzyme is inhibited by Zn 2؉ and succinylacetone. The presence of PfALAD in P. falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy. The enzyme has been localized to the apicoplast of the malaria parasite. Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium. It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg 2؉ independence for catalytic activity or octamer stabilization. Expression levels of PfALAD in P. falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite. It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite.Studies in this laboratory had demonstrated that the malaria parasites (Plasmodium falciparum and Plasmodium berghei) are capable of heme biosynthesis de novo, despite acquiring large amounts of heme from the host red cell hemoglobin in the intraerythrocytic stage (1, 2). Inhibition of the de novo heme biosynthetic pathway leads to death of the parasite and the pathway is therefore a drug target (1-3). Studies on the enzymes of the heme-biosynthetic pathway have revealed that the first enzyme, ␦-aminolevulinate synthase, is coded for by the parasite genome and is localized in the parasite mitochondrion (4, 5). Studies from this laboratory have shown that the second enzyme of the pathway, ␦-aminolevulinate dehydratase (ALAD), 1 in the parasite is of host origin and evidence was provided to show that the enzyme is translocated (imported) from the host red cell into the parasite and is functional (2, 3).At the same time genome sequence data available for P. falciparum indicates that the parasite genome codes for ALAD (PfALAD) and other enzymes of the heme-biosynthetic pathway. Sato et al. (6) have shown by phylogenetic amino acid sequence analysis derived from truncated...

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Section: Localization Of Alad In the Parasitementioning

confidence: 86%

δ-Aminolevulinic Acid Dehydratase from Plasmodium falciparum

Dhanasekaran

Chandra

Sagar

et al. 2004

Journal of Biological Chemistry

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“…HDEL) (Hager et al, 1999). (4) Construction methods have previously been described for the basic GFP-expressing vector pdhfrΔCAT-GFP, and the secretion vectors ptubP30 L -GFP and ptubP30 L -YFP Matrajt et al, 2002), as well as constructs that label the ER (ptubP30 L -GFP-HDEL) (Hager et al, 1999), Golgi (ptubGRASP-YFP) (Pelletier et al, 2002), IMC (ptubIMC1-YFP) , micronemes (ptubMIC3-GFP) , rhoptries (ptubROP1-CAT-YFP) (Dzierszinski et al, 2004) and apicoplast [ptubACP L -EGFP (Waller et al, 1998); ptubFNR L -YFP (Striepen et al, 2000;Harb et al, 2004); ptubFNR L -DsRed (He et al, 2001)]. For labeling the mitochondrion, sequences encoding the 55 N-terminal residues of T. gondii heatshock protein 60 (Toursel et al, 2000) were amplified from tachyzoite cDNA using primers 5Ј-atgcagatctaaaATGcttgcccgcgcttca-3Ј and 5Ј-cagtcctagggccgagagtgactccgac-3Ј, and introduced as a BglII-AvrII fragment into ptubIMC1-YFP and ptubFNR L -DsRed, yielding ptubHSP60 L -YFP and ptubHSP60 L -DsRed, respectively.…”

Section: Molecular Methodsmentioning

confidence: 99%

“…Fluorescent markers greatly facilitate the study of organellar dynamics in living parasites Striepen et al, 2000;He et al, 2001;Hu et al, 2002;Pelletier et al, 2002;Dzierszinski et al, 2004;Vaishnava et al, 2005;van Dooren et al, 2005;Hartmann et al, 2006;Hu et al, 2006). For example, the parasite Golgi has been shown to elongate laterally before dividing and segregating between the two daughter cells (Pelletier et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Organellar dynamics during the cell cycle of Toxoplasma gondii

Nishi

Murray

et al. 2008

Journal of Cell Science

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249

341

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results in partitioning of the apicoplast, nucleus, endoplasmic reticulum, and finally the mitochondrion, which enters the developing daughters rapidly, but only very late during the division cycle. The specialized secretory organelles (micronemes and rhoptries) form de novo. This distinctive pattern of replication -in which organellar segregation spans ~75% of the cell cycle, completely encompassing S phase -suggests an unusual mechanism of cell cycle regulation. Supplementary material available online at

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“…gondii uses the same general mechanism to mediate transport of proteins into the apicoplast as Plasmodium (He et al 2001). A bipartite leader peptide, including a primary secretory domain followed by a secondary plastid transit domain, can be predicted by specialized tools including PlasmoAP (Foth et al 2003) and PATS (Waller et al 1998 ;Zuegge et al 2001) in PlasmoDB.…”

Section: Prediction Of Localization Of Tgprex In Silicomentioning

confidence: 99%

The Toxoplasma gondii Plastid replication and Repair Enzyme Complex, PREX

et al. 2009

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A plastid-like organelle, the apicoplast, is essential to the majority of medically and veterinary important apicomplexan protozoa including Toxoplasma gondii and Plasmodium. The apicoplast contains multiple copies of a 35 kb genome, the replication of which is dependent upon nuclear-encoded proteins that are imported into the organelle. In P. falciparum an unusual multi-functional gene, pfprex, was previously identified and inferred to encode a protein with DNA primase, DNA helicase and DNA polymerase activities. Herein, we report the presence of a prex orthologue in T. gondii. The protein is predicted to have a bi-partite apicoplast targeting sequence similar to that demonstrated on the PfPREX polypeptide, capable of delivering marker proteins to the apicoplast. Unlike the P. falciparum gene that is devoid of introns, the T. gondii prex gene carries 19 introns, which are spliced to produce a contiguous mRNA. Bacterial expression of the polymerase domain reveals the protein to be active. Consistent with the reported absence of a plastid in Cryptosporidium species, in silico analysis of their genomes failed to demonstrate an orthologue of prex. These studies indicate that prex is conserved across the plastid-bearing apicomplexans and may play an important role in the replication of the plastid genome.

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Targeting and Processing of Nuclear-encoded Apicoplast Proteins in Plastid Segregation Mutants of Toxoplasma gondii

Cited by 52 publications

References 43 publications

δ-Aminolevulinic Acid Dehydratase from Plasmodium falciparum

δ-Aminolevulinic Acid Dehydratase from Plasmodium falciparum

Organellar dynamics during the cell cycle of Toxoplasma gondii

The Toxoplasma gondii Plastid replication and Repair Enzyme Complex, PREX

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