Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting

DeRocher, Amy E.; Gilbert, Brian E.; Feagin, Jean E.; Parsons, Marilyn

doi:10.1242/jcs.01627

Cited by 61 publications

(95 citation statements)

References 41 publications

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“…Recovery of the fluorescent signal was observed in both S. neurona and T. gondii plastids over a period of 1 hour (data not shown). Signal recovery probably occurred by import and is consistent with the kinetics of apicoplast targeting in P. falciparum (van Dooren et al, 2002) and T. gondii (Derocher et al, 2005).…”

Section: Resultssupporting

confidence: 76%

Plastid segregation and cell division in the apicomplexan parasiteSarcocystis neurona

Vaishnava

Morrison

Gaji

et al. 2005

Journal of Cell Science

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Apicomplexan parasites harbor a secondary plastid that is essential to their survival. Several metabolic pathways confined to this organelle have emerged as promising parasite-specific drug targets. The maintenance of the organelle and its genome is an equally valuable target. We have studied the replication and segregation of this important organelle using the parasite Sarcocystis neurona as a cell biological model. This model system makes it possible to differentiate and dissect organellar growth, fission and segregation over time, because of the parasite's peculiar mode of cell division. S. neurona undergoes five cycles of chromosomal replication without nuclear division, thus yielding a cell with a 32N nucleus. This nucleus undergoes a sixth replication cycle concurrent with nuclear division and cell budding to give rise to 64 haploid daughter cells. Interestingly, intranuclear spindles persist throughout the cell cycle, thereby providing a potential mechanism to organize chromosomes and organelles in an organism that undergoes dramatic changes in ploidy. The development of the plastid mirrors that of the nucleus, a continuous organelle, which grows throughout the parasite's development and shows association with all centrosomes. Pharmacological ablation of the parasite's multiple spindles demonstrates their essential role in the organization and faithful segregation of the plastid. By using several molecular markers we have timed organelle fission to the last replication cycle and tied it to daughter cell budding. Finally, plastids were labeled by fluorescent protein expression using a newly developedS. neurona transfection system. With these transgenic parasites we have tested our model in living cells employing laser bleaching experiments.

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Section: Resultssupporting

confidence: 76%

Plastid segregation and cell division in the apicomplexan parasiteSarcocystis neurona

Vaishnava

Morrison

Gaji

et al. 2005

Journal of Cell Science

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“…The exploitation of a ligand-regulated system that allows the accumulation and release of proteins from the ER (68) generated more convincing conclusions. Ligand-induced release of a NEAT reporter from the ER led to rapid localization to the apicoplast, which was not blocked by brefeldin A (14). Furthermore, low-temperature incubation, which blocks Golgi trafficking, did not block the apicoplast localization of the reporter upon ligand-induced release.…”

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confidence: 88%

“…Several experiments argue against the Golgi model. First, the treatment of parasites with the Golgi inhibitor brefeldin A does not lead to a change in the steady-state distribution of NEAT reporters in either T. gondii (14) or P. falciparum (81). The exploitation of a ligand-regulated system that allows the accumulation and release of proteins from the ER (68) generated more convincing conclusions.…”

mentioning

confidence: 99%

“…Furthermore, cleavage of the transit peptide is quite delayed compared to the time of synthesis (14,84). The lag in transit peptide processing could represent inefficient cleavage of the imported protein or a lengthy time course for import across all membranes (because of a generally inefficient import process or perhaps cell cycle regulation of certain steps).…”

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confidence: 99%

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Protein Trafficking to the Apicoplast: Deciphering the Apicomplexan Solution to Secondary Endosymbiosis

et al. 2007

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“…Trafficking occurs via the secretory pathway and is guided by an N-terminal bipartite targeting sequence (9). Transport from the ER to the apicoplast is believed to be vesicle mediated and to sidestep the Golgi apparatus (10,11). Once delivered to the apicoplast, proteins have to cross three additional membranes.…”

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confidence: 99%

Genetic Evidence that an Endosymbiont-derived Endoplasmic Reticulum-associated Protein Degradation (ERAD) System Functions in Import of Apicoplast Proteins

Agrawal

Dooren

Beatty

et al. 2009

Journal of Biological Chemistry

173

229

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Most apicomplexan parasites harbor a relict chloroplast, the apicoplast, that is critical for their survival. Whereas the apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the apicoplast. We engineered a conditional null mutant in apicoplast Der1, the putative pore of the apicoplast ERAD complex, and found that loss of Der1 Ap results in loss of apicoplast protein import and subsequent death of the parasite.Apicomplexa are a phylum of obligate parasites that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Recent evidence suggests that apicomplexans evolved from a free-living photosynthetic ancestor (1). This ancestry is reflected in the presence of a chloroplast-like organelle, the apicoplast (2). While no longer engaged in photosynthesis, the apicoplast is essential to parasite survival and home to several critical biosynthetic pathways. Bioinformatic and experimental evidence suggests that the apicoplast is engaged in the synthesis of fatty acids, isoprenoids, and heme (3, 4). Genetic or pharmacological ablation of these pathways blocks parasite growth and the apicoplast, therefore, is currently considered a prime target for antiparasitic drug development (5-7). The organelle was derived by secondary endosymbiosis and reflects the successful union of a red alga and a heterotrophic eukaryote (8). A large number of algal genes were transferred to the host nucleus, and their products must now be routed back into the organelle. Trafficking occurs via the secretory pathway and is guided by an N-terminal bipartite targeting sequence (9). Transport from the ER to the apicoplast is believed to be vesicle mediated and to sidestep the Golgi apparatus (10, 11). Once delivered to the apicoplast, proteins have to cross three additional membranes. Recently, we demonstrated that a homolog of Tic20, a component of the translocon of the inner chloroplast membrane (Tic) 4 complex in plants, is likely required for protein import across the innermost membrane of the apicoplast (12). To date bioinformatics searches have failed to identify a matching translocon of the outer chloroplast membrane in apicomplexans (13). Analysis of the genome of the remnant nucleus of the algal endosymbiont in cryptomonads showed the presence of an ER-associated protein degradation (ERAD) system and offered a candidate for a translocon across the third and potentially the second membrane (14). Classically, ERAD functions in the homeostasis of the secretory ...

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Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting

Cited by 61 publications

References 41 publications

Plastid segregation and cell division in the apicomplexan parasiteSarcocystis neurona

Plastid segregation and cell division in the apicomplexan parasiteSarcocystis neurona

Protein Trafficking to the Apicoplast: Deciphering the Apicomplexan Solution to Secondary Endosymbiosis

Genetic Evidence that an Endosymbiont-derived Endoplasmic Reticulum-associated Protein Degradation (ERAD) System Functions in Import of Apicoplast Proteins

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