Biology subjects

Maclean, A. E.

Publications and source records attributed to Maclean, A. E..

9 recordsLinked to original sources

bioRxiv2026

Doxycycline inhibits both apicoplast and mitochondrial translation in apicomplexan parasites

Doxycycline is a tetracycline-class antibiotic used for malarial prophylaxis and as an occasional partner drug in malaria treatment. Doxycyclines antimalarial mechanism of action is attributed to inhibiting the prokaryotic 70S ribosomes of the apicoplast, a non-photosynthetic plastid present in most apicomplexan species including Plasmodium and Toxoplasma. At lower concentrations (<5 M) doxycycline exhibits a delayed death phenotype, typical of inhibitors of apicoplast housekeeping processes. Unlike other delayed death drugs (e.g. clindamycin), doxycycline has rapid schizonticidal activity at higher concentrations (>10 M) via an unknown and apicoplast-independent mechanism. In other eukaryotes, and plausibly in Plasmodium and Toxoplasma, doxycycline inhibits mitochondrial 70S ribosomes. Here we use a mass spectrometry approach to assess steady state and turnover for apicoplast DNA encoded proteins in Plasmodium falciparum. We directly show that these proteins decrease in both abundance and synthesis following treatment with doxycycline and clindamycin. High concentrations of doxycycline, but not clindamycin, also reduced the abundance of a mitochondrial-encoded protein. Proteins encoded by the mitochondrial genome are required for the formation of complexes III and IV in the electron transport chain. We show a reduction in abundance and activity of complex IV following doxycycline treatment in Toxoplasma gondii. High doxycycline concentrations also disrupt oxidative phosphorylation in both P. falciparum and T. gondii, likely as a consequence of impaired complex III and IV formation. For the first time we directly measure apicoplast translation inhibition and characterise doxycycline as a mitochondrial translation inhibitor of P. falciparum and T. gondii.

microbiology↗

bioRxiv2025

Complexome profile of mitochondrial complexes in the myzozoan parasite of oysters, Perkinsus marinus

Mitochondrial complexes, such as the mitochondrial electron transport chain (mETC), the F1Fo-ATP synthase and the mitochondrial ribosome, are centrally important for mitochondrial function. Recently, an unexpected diversity in the composition of these complexes across unicellular eukaryotic lineages, with the apicomplexan parasites featuring prominently, has been revealed. However, whether the observed enlarged and divergent mitochondrial complexes are conserved across the wider Myzozoan lineage has not been investigated. Here, using a complexome profiling proteomic approach, we have characterised the composition of the mitochondrial complexes of the myzozoan parasite of oysters, Perkinsus marinus. We show that it shares with Plasmodium and Toxoplasma huge ATP synthase and mETC complexes. Moreover, the Perkinsus mitoribosome possesses many of the divergent features recently discovered in apicomplexans, such as an expanded subunit repertoire and the inclusion of RAP-domain and ApiAP2-like proteins as ribosomal proteins. Our study reveals the divergent subunit composition of mitochondrial complexes is an ancestral and highly conserved feature of Myzozoa.

microbiology↗

bioRxiv2024

The Toxoplasma gondii mitochondrial transporter ABCB7 is essential for cytosolic iron-sulfur cluster biogenesis and protein translation

Iron-sulfur (Fe-S) clusters are ubiquitous inorganic cofactors required for numerous essential cellular pathways. Since they cannot be scavenged from the environment, Fe-S clusters are synthesised de novo in cellular compartments such as the apicoplast, mitochondrion and cytosol. The cytosolic Fe-S cluster biosynthesis pathway relies on transport of an intermediate from the mitochondrial pathway. An ATP binding cassette (ABC) transporter called ABCB7 is responsible for this role in numerous commonly studied organisms, but its role in the medically important apicomplexan parasites has not yet been studied. Here we identify and characterise the Toxoplasma gondii ABCB7 homolog. Genetic depletion shows that it is essential for parasite growth, and that disruption triggers partial stage conversion. Characterisation of the knock-down line highlights a defect in cytosolic Fe-S cluster biogenesis leading to defects in protein translation and other pathways including DNA and RNA replication and metabolism. Our work provides support for a broad conservation of the connection between mitochondrial and cytosolic in Fe-S cluster biosynthesis and reveal its importance for parasite survival.

microbiology↗

bioRxiv2021

The Fe-S cluster assembly factors NFU4 and NFU5 are primarily required for protein lipoylation in mitochondria

Plants have evolutionarily conserved NFU-domain proteins that are targeted to plastids or mitochondria. The plastid-type NFU1, NFU2 and NFU3 in Arabidopsis thaliana play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here we confirm that NFU4 and NFU5 are targeted to the mitochondria. The proteins are constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of the proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes and aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth was improved by elevated CO2. In addition, pyruvate, 2-oxoglutarate and branched-chain amino acids accumulated in the nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, in providing Fe-S clusters to lipoyl synthase. One sentence summaryA pair of evolutionarily conserved proteins involved in iron-sulfur cofactor assembly have a specific role in lipoate biosynthesis for mitochondrial dehydrogenases.

plant biology↗

bioRxiv2020

Complexome profile of Toxoplasma gondii mitochondria identifies a divergent cytochrome bc1 complex

The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Here, using a complexome profiling proteomic approach, we have characterised the Toxoplasma mETC complexes and F1Fo-ATP synthase. We identified and assigned 60 proteins to complexes II, IV and F1Fo-ATP synthase of Toxoplasma, of which 16 have not been identified previously. Notably, our complexome profile elucidates the composition of the Toxoplasma complex III, the target of clinically used drugs such as atovaquone. We identified two new homologous subunits and two new parasite-specific subunits, one of which is broadly conserved in myzozoans. We demonstrate all four proteins are essential for complex III stability and parasite growth, and show their depletion leads to decreased mitochondrial potential, supporting their role as complex III subunits. Our study highlights the divergent subunit composition of the apicomplexan mETC complexes and sets the stage for future structural and drug discovery studies. Author SummaryApicomplexan parasites, such as Toxoplasma and Plasmodium, cause diseases of global importance, such as toxoplasmosis and malaria. The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase, which provide the parasite with energy and important metabolites, are essential for parasite function. Here, using a proteomic technique called complexome profiling, we report the composition of the Toxoplasma mETC and F1Fo-ATP synthase. In particular, we reveal the compositions of complexes II and III for the first time. Complex III is an important drug target, yet its full protein composition was unknown. We identify new parasite-specific complex III subunits and demonstrate that they are essential for parasite survival and for proper functioning of the mETC. Our study highlights the divergent nature of the apicomplexan mETC and F1Fo-ATP synthase.

cell biology↗

bioRxiv2020

Branched-chain amino acid catabolism depends on GRXS15 through mitochondrial lipoyl cofactor homeostasis

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol and mitochondria. A single monothiol glutaredoxin (GRX) has been shown to be involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homologue GRXS15 has only partially been characterized. Arabidopsis grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype. In an in-depth metabolic analysis, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis and the electron transport chain. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, 2-oxoglutarate, glycine and branched-chain amino acids (BCAAs). The most pronounced accumulation occurred in branched-chain -keto acids (BCKAs), the first degradation products resulting from deamination of BCAAs. In wild-type plants, pyruvate, 2-oxoglutarate, glycine and BCKAs are all metabolized through decarboxylation by four mitochondrial lipoyl cofactor-dependent dehydrogenase complexes. Because these enzyme complexes are very abundant and the biosynthesis of the lipoyl cofactor depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why lipoyl cofactor-dependent processes are most sensitive to restricted Fe-S supply in GRXS15 K83A mutants. One-sentence summaryDeficiency in GRXS15 restricts protein lipoylation and causes metabolic defects in lipoyl cofactor-dependent dehydrogenase complexes, with branched-chain amino acid catabolism as dominant bottleneck.

plant biology↗

bioRxiv2018

Pathogenic mutations in NUBPL affect complex I activity and cold tolerance in the yeast model Yarrowia lipolytica

Complex I deficiency is a common cause of mitochondrial disease, resulting from mutations in genes encoding structural subunits, assembly factors or defects in mitochondrial gene expression. Advances in genetic diagnostics and sequencing have led to identification of several variants in NUBPL, an assembly factor of complex I, which are potentially pathogenic. To help assign pathogenicity and learn more about the function of NUBPL, amino acid substitutions were recreated in the homologous Ind1 protein of the yeast model Yarrowia lipolytica. L102P destabilized the Ind1 protein, leading to a null-mutant phenotype. D103Y, L191F and G285C affected complex I assembly to varying degrees, whereas the G138D variant did not impact on complex I levels or dNADH:ubiquinone activity. Blue-native PAGE and immunolabelling of the structural subunits NUBM and NUCM revealed that all Ind1 variants accumulated a Q-module intermediate of complex I. In the D103Y variant the matrix arm intermediate was virtually absent, indicating a dominant effect. Dysfunction of Ind1, but not absence of complex I, rendered Y. lipolytica sensitive to cold. The Ind1 G285C variant was able to support complex I assembly at 28{degrees}C, but not at 10{degrees}C. Our results indicate that Ind1 is required for progression of assembly from the Q module to the full matrix arm. Cold sensitivity could be developed as a phenotype assay to demonstrate pathogenicity of NUBPL mutations and other complex I defects.

biochemistry↗

bioRxiv2018

Vacuolar iron stores gated by NRAMP3 and NRAMP4 are the primary source of iron in germinating seeds

During seed germination, iron (Fe) stored in vacuoles is exported by the redundant NRAMP3 and NRAMP4 transporter proteins. A double nramp3 nramp4 mutant is unable to mobilize Fe stores and does not develop in the absence of external Fe. We used RNA sequencing to compare gene expression in nramp3 nramp4 and wild type during germination and early seedling development. Even though sufficient Fe was supplied, the Fe-responsive transcription factors bHLH38, 39, 100 and 101 and their downstream targets FRO2 and IRT1 mediating Fe uptake were strongly upregulated in the nramp3 nramp4 mutant. Activation of the Fe deficiency response was confirmed by increased ferric chelate reductase activity in the mutant. At early stages, genes important for chloroplast redox control (FSD1, SAPX), Fe homeostasis (FER1, SUFB) and chlorophyll metabolism (HEMA1, NYC1) were downregulated, indicating limited Fe availability in plastids. In contrast, expression of FRO3, encoding a ferric reductase involved in Fe import into the mitochondria, was maintained and Fe-dependent enzymes in the mitochondria were unaffected in nramp3 nramp4. Together these data show that a failure to mobilize Fe stores during germination triggered Fe deficiency responses and strongly affected plastids but not mitochondria.

plant biology↗

bioRxiv2017

Genetic dissection of cyclic pyranopterin monophosphate biosynthesis in plant mitochondria

Mitochondria play a key role in the biosynthesis of two metal cofactors, iron-sulfur (FeS) clusters and molybdenum cofactor (Moco). The two pathways intersect at several points, but a scarcity of mutants has hindered studies to better understand these links. We screened a collection of sirtinol-resistant Arabidopsis thaliana mutants for lines with decreased activities of cytosolic FeS enzymes and Moco enzymes. We identified a new mutant allele of ATM3, encoding the ATP-binding cassette Transporter of the Mitochondria 3 (systematic name ABCB25), confirming the previously reported role of ATM3 in both FeS cluster and Moco biosynthesis. We also identified a mutant allele in CNX2, Cofactor of Nitrate reductase and Xanthine dehydrogenase 2, encoding GTP 3',8-cyclase, the first step in Moco biosynthesis which is localized in the mitochondria. A single nucleotide polymorphism in cnx2-2 leads to substitution of Arg88 with Gln in the N-terminal FeS cluster-binding motif. cnx2-2 plants are small and chlorotic, with severely decreased Moco enzyme activities, but they performed better than a cnx2-1 knockout mutant, which could only survive with ammonia as nitrogen source. Measurement of cyclic pyranopterin monophosphate (cPMP) levels by LC-MS/MS showed that this Moco intermediate was below the limit of detection in both cnx2-1 and cnx2-2, and accumulated more than 10-fold in seedlings mutated in the downstream gene CNX5. Interestingly, atm3-1 mutants had less cPMP than wild type, correlating with previous reports of a similar decrease in nitrate reductase activity. Taken together, our data functionally characterise CNX2 and suggest that ATM3 is indirectly required for cPMP synthesis.

biochemistry↗