Identification of cryptic subunits from an apicomplexan ATP synthase
TL;DR: 11 previously unknown subunits from the Toxoplasma ATP synthase are identified, which lack homologs outside the phylum, and highlight divergent features of the central metabolic machinery in apicomplexans, which may reveal new therapeutic opportunities.
Abstract: The mitochondrial ATP synthase is a macromolecular motor that uses the proton gradient to generate ATP. Proper ATP synthase function requires a stator linking the catalytic and rotary portions of the complex. However, sequence-based searches fail to identify genes encoding stator subunits in apicomplexan parasites like Toxoplasma gondii or the related organisms that cause malaria. Here, we identify 11 previously unknown subunits from the Toxoplasma ATP synthase, which lack homologs outside the phylum. Modeling suggests that two of them, ICAP2 and ICAP18, are distantly related to mammalian stator subunits. Our analysis shows that both proteins form part of the ATP synthase complex. Depletion of ICAP2 leads to aberrant mitochondrial morphology, decreased oxygen consumption, and disassembly of the complex, consistent with its role as an essential component of the Toxoplasma ATP synthase. Our findings highlight divergent features of the central metabolic machinery in apicomplexans, which may reveal new therapeutic opportunities.
Citations
More filters
01 Sep 2016
TL;DR: The first genome-wide genetic screen of an apicomplexan parasite is presented, revealing essential functions during infection of human cells and providing broad-based functional information on T. gondii genes that will facilitate future approaches to expand the horizon of antiparasitic interventions.
Abstract: National Institute of General Medical Sciences (U.S.) (Center for Integrative Synthetic Biology Grant P50GM098792)
456 citations
TL;DR: This study identifies approximately 400 mitochondrial proteins, many of which lack homologs in the animals that these parasites infect, and most of which are important for parasite growth, and demonstrates that one such protein, termed TgApiCox25, is an important component of the parasite cytochrome c oxidase (COX) complex.
Abstract: The mitochondrion of apicomplexan parasites is critical for parasite survival, although the full complement of proteins that localize to this organelle has not been defined Here we undertake two independent approaches to elucidate the mitochondrial proteome of the apicomplexan Toxoplasma gondii We identify approximately 400 mitochondrial proteins, many of which lack homologs in the animals that these parasites infect, and most of which are important for parasite growth We demonstrate that one such protein, termed TgApiCox25, is an important component of the parasite cytochrome c oxidase (COX) complex We identify numerous other apicomplexan-specific components of COX, and conclude that apicomplexan COX, and apicomplexan mitochondria more generally, differ substantially in their protein composition from the hosts they infect Our study highlights the diversity that exists in mitochondrial proteomes across the eukaryotic domain of life, and provides a foundation for defining unique aspects of mitochondrial biology in an important phylum of parasites
84 citations
TL;DR: In this paper, the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae, which is held together by interactions between parasite-specific subunits in the lumenal region.
Abstract: Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit-a. The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF1. Cryo-ET and subtomogram averaging revealed that hexamers arrange into ~20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa.
53 citations
TL;DR: This article applied complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum and found that respiratory chain complex components and linked metabolic pathways are up to 40 times more prevalent in gametocytes, while glycolytic enzymes are substantially reduced.
Abstract: Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.
34 citations
TL;DR: This paper applied complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum and found that respiratory chain complex components and linked metabolic pathways are up to 40 times more prevalent in gametocytes, while glycolytic enzymes are substantially reduced.
Abstract: Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.
33 citations
Cites background from "Identification of cryptic subunits ..."
...Recent studies in the related apicomplexan parasite 46 Toxoplasma gondii have suggested a divergent and unusual composition of cytochrome c oxidase (CIV; 47 (10) and F1FO-ATP synthase (CV)(11, 12)....
[...]
References
More filters
TL;DR: The Clustal W and ClUSTal X multiple sequence alignment programs have been completely rewritten in C++ to facilitate the further development of the alignment algorithms in the future and has allowed proper porting of the programs to the latest versions of Linux, Macintosh and Windows operating systems.
Abstract: Summary: The Clustal W and Clustal X multiple sequence alignment programs have been completely rewritten in C++. This will facilitate the further development of the alignment algorithms in the future and has allowed proper porting of the programs to the latest versions of Linux, Macintosh and Windows operating systems.
Availability: The programs can be run on-line from the EBI web server: http://www.ebi.ac.uk/tools/clustalw2. The source code and executables for Windows, Linux and Macintosh computers are available from the EBI ftp site ftp://ftp.ebi.ac.uk/pub/software/clustalw2/
Contact: clustalw@ucd.ie
25,325 citations
"Identification of cryptic subunits ..." refers methods in this paper
...Alignment was performed using ClustalW (Larkin et al., 2007), and the phylogenetic tree was generated by neighbor-joining excluding positions with gaps....
[...]
TL;DR: Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining, and this work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
Abstract: Proteins from silver-stained gels can be digested enzymatically and the resulting peptides analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nanoelectrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.
8,437 citations
"Identification of cryptic subunits ..." refers methods in this paper
...…beads for 1 hr at 4˚C, and elution was carried out by adding a solution containing 150 ng/ml of Ty peptide diluted in IP lysis buffer to the beads, followed by a 30 min incubation at 4˚C. Proteins were resolved by SDS-PAGE and visualized by silver stain as described in (Shevchenko et al., 1996)....
[...]
TL;DR: An updated protocol for Phyre2, which uses advanced remote homology detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants for a user's protein sequence.
Abstract: Phyre2 is a web-based tool for predicting and analyzing protein structure and function. Phyre2 uses advanced remote homology detection methods to build 3D models, predict ligand binding sites, and analyze amino acid variants in a protein sequence. Phyre2 is a suite of tools available on the web to predict and analyze protein structure, function and mutations. The focus of Phyre2 is to provide biologists with a simple and intuitive interface to state-of-the-art protein bioinformatics tools. Phyre2 replaces Phyre, the original version of the server for which we previously published a paper in Nature Protocols. In this updated protocol, we describe Phyre2, which uses advanced remote homology detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants (e.g., nonsynonymous SNPs (nsSNPs)) for a user's protein sequence. Users are guided through results by a simple interface at a level of detail they determine. This protocol will guide users from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain composition and model quality. A range of additional available tools is described to find a protein structure in a genome, to submit large number of sequences at once and to automatically run weekly searches for proteins that are difficult to model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2
. A typical structure prediction will be returned between 30 min and 2 h after submission.
7,941 citations
"Identification of cryptic subunits ..." refers background in this paper
...Pattern hidden Markov models and secondary structure predictions—implemented in HHPRED or Phyre2 (Kelley et al., 2015; Zimmermann et al., 2018)—first hinted at the homology between ICAP2 and the b subunit of the mammalian ATP synthase....
[...]
TL;DR: A neural network-based tool, TargetP, for large-scale subcellular location prediction of newly identified proteins has been developed and it is estimated that 10% of all plant proteins are mitochondrial and 14% chloroplastic, and that the abundance of secretory proteins, in both Arabidopsis and Homo, is around 10%.
4,268 citations
"Identification of cryptic subunits ..." refers methods in this paper
...Subcellular localization predictions were performed with iPSORT and TargetP 1.1 (Emanuelsson et al., 2000)....
[...]
TL;DR: In their Perspective, Hoppins and Nunnari explain that the endoplasmic reticulum is an active participant in mitochondrial division and discuss how mitochondrial dynamics and cell death are linked.
Abstract: Mitochondrial fission and fusion play critical roles in maintaining functional mitochondria when cells experience metabolic or environmental stresses. Fusion helps mitigate stress by mixing the contents of partially damaged mitochondria as a form of complementation. Fission is needed to create new mitochondria, but it also contributes to quality control by enabling the removal of damaged mitochondria and can facilitate apoptosis during high levels of cellular stress. Disruptions in these processes affect normal development, and they have been implicated in neurodegenerative diseases, such as Parkinson’s.
2,560 citations