Dataset Information

The HU protein is important for apicoplast genome maintenance and inheritance in Toxoplasma gondii.

ABSTRACT: The apicoplast, a chloroplast-like organelle, is an essential cellular component of most apicomplexan parasites, including Plasmodium and Toxoplasma. The apicoplast maintains its own genome, a 35-kb DNA molecule that largely encodes proteins required for organellar transcription and translation. Interference with apicoplast genome maintenance and function is a validated target for drug therapy for malaria and toxoplasmosis. However, the many proteins required for genome maintenance and inheritance remain largely unstudied. Here we genetically characterize a nucleus-encoded homolog to the bacterial HU protein in Toxoplasma gondii. In bacteria, HU is a DNA-binding structural protein with fundamental roles in transcription, replication initiation, and DNA repair. Immunofluorescence assays reveal that in T. gondii this protein localizes to the apicoplast. We have found that the HU protein from Toxoplasma can successfully complement bacterial ?hupA mutants, supporting a similar function. We were able to construct a genetic knockout of HU in Toxoplasma. This ?hu mutant is barely viable and exhibits significant growth retardation. Upon further analysis of the mutant phenotype, we find that this mutant has a dramatically reduced apicoplast genome copy number and, furthermore, suffers defects in the segregation of the apicoplast organelle. Our findings not only show that the HU protein is important for Toxoplasma cell biology but also demonstrate the importance of the apicoplast genome in the biogenesis of the organelle.

SUBMITTER: Reiff SB

PROVIDER: S-EPMC3416497 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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The HU protein is important for apicoplast genome maintenance and inheritance in Toxoplasma gondii.

Reiff Sarah B SB Vaishnava Shipra S Striepen Boris B

Eukaryotic cell 20120518 7

The apicoplast, a chloroplast-like organelle, is an essential cellular component of most apicomplexan parasites, including Plasmodium and Toxoplasma. The apicoplast maintains its own genome, a 35-kb DNA molecule that largely encodes proteins required for organellar transcription and translation. Interference with apicoplast genome maintenance and function is a validated target for drug therapy for malaria and toxoplasmosis. However, the many proteins required for genome maintenance and inheritan ...[more]

PMID: 22611021

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Project description:UNLABELLED:Autophagy is a catabolic process widely conserved among eukaryotes that permits the rapid degradation of unwanted proteins and organelles through the lysosomal pathway. This mechanism involves the formation of a double-membrane structure called the autophagosome that sequesters cellular components to be degraded. To orchestrate this process, yeasts and animals rely on a conserved set of autophagy-related proteins (ATGs). Key among these factors is ATG8, a cytoplasmic protein that is recruited to nascent autophagosomal membranes upon the induction of autophagy. Toxoplasma gondii is a potentially harmful human pathogen in which only a subset of ATGs appears to be present. Although this eukaryotic parasite seems able to generate autophagosomes upon stresses such as nutrient starvation, the full functionality and biological relevance of a canonical autophagy pathway are as yet unclear. Intriguingly, in T. gondii, ATG8 localizes to the apicoplast under normal intracellular growth conditions. The apicoplast is a nonphotosynthetic plastid enclosed by four membranes resulting from a secondary endosymbiosis. Using superresolution microscopy and biochemical techniques, we show that TgATG8 localizes to the outermost membrane of this organelle. We investigated the unusual function of TgATG8 at the apicoplast by generating a conditional knockdown mutant. Depletion of TgATG8 led to rapid loss of the organelle and subsequent intracellular replication defects, indicating that the protein is essential for maintaining apicoplast homeostasis and thus for survival of the tachyzoite stage. More precisely, loss of TgATG8 led to abnormal segregation of the apicoplast into the progeny because of a loss of physical interactions of the organelle with the centrosomes. IMPORTANCE:By definition, autophagy is a catabolic process that leads to the digestion and recycling of eukaryotic cellular components. The molecular machinery of autophagy was identified mainly in model organisms such as yeasts but remains poorly characterized in phylogenetically distant apicomplexan parasites. We have uncovered an unusual function for autophagy-related protein ATG8 in Toxoplasma gondii: TgATG8 is crucial for normal replication of the parasite inside its host cell. Seemingly unrelated to the catabolic autophagy process, TgATG8 associates with the outer membrane of the nonphotosynthetic plastid harbored by the parasite called the apicoplast, and there it plays an important role in the centrosome-driven inheritance of the organelle during cell division. This not only reveals an unexpected function for an autophagy-related protein but also sheds new light on the division process of an organelle that is vital to a group of important human and animal pathogens.

Project description:Apicomplexan parasites cause a variety of important infectious diseases, including malaria, toxoplasma encephalitis, and severe diarrhea due to Cryptosporidium Most apicomplexans depend on an organelle called the apicoplast which is derived from a red algal endosymbiont. The apicoplast is essential for the parasite as the compartment of fatty acid, heme, and isoprenoid biosynthesis. The majority of the approximate 500 apicoplast proteins are nucleus encoded and have to be imported across the four membranes that surround the apicoplast. Import across the second outermost membrane of the apicoplast, the periplastid membrane, depends on an apicoplast-specific endoplasmic reticulum-associated protein degradation (ERAD) complex and on enzymes of the associated ubiquitination cascade. However, identification of an apicoplast ubiquitin associated with this machinery has long been elusive. Here we identify a plastid ubiquitin-like protein (PUBL), an apicoplast protein that is derived from a ubiquitin ancestor but that has significantly changed in its primary sequence. PUBL is distinct from known ubiquitin-like proteins, and phylogenomic analyses suggest a clade specific to apicomplexans. We demonstrate that PUBL and the AAA ATPase CDC48AP both act to translocate apicoplast proteins across the periplastid membrane during protein import. Conditional null mutants and genetic complementation show that both proteins are critical for this process and for parasite survival. PUBL residues homologous to those that are required for ubiquitin conjugation onto target proteins are essential for this function, while those required for polyubiquitination and preprotein processing are dispensable. Our experiments provide a mechanistic understanding of the molecular machinery that drives protein import across the membranes of the apicoplast.IMPORTANCE Apicomplexan parasites are responsible for important human diseases. There are no effective vaccines for use in humans, and drug treatment faces multiple challenges, including emerging resistance, lack of efficacy across the lifecycle, and adverse drug effects. The apicoplast is a promising target for novel treatments: this chloroplast-like organelle is derived from an algal symbiont, is absent from the host, and is essential for parasite growth and pathogenesis. We use Toxoplasma gondii as a model to study the apicoplast due to its strong genetic tools and established functional assays. We identify a plastid ubiquitin-like protein (PUBL) which is a novel ubiquitin-like protein and demonstrate its importance and that of the motor protein CDC48AP for apicoplast protein import. These findings broaden our understanding of the evolution and mechanistic workings of a unique parasite organelle and may lead to new opportunities for treatments against important human pathogens.

Dataset Information

The HU protein is important for apicoplast genome maintenance and inheritance in Toxoplasma gondii.

Publications

The HU protein is important for apicoplast genome maintenance and inheritance in Toxoplasma gondii.

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