Divergent acyl carrier protein decouples mitochondrial Fe-S cluster biogenesis from fatty acid synthesis in malaria parasites
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Evaluation Summary:
This study defines the role of a divergent mitochondrial-localized isoform of a FASII acyl carrier protein (mACP) in the malaria parasite, Plasmodium falciparum. In contrast to the situation in other eukaryotes, mACP is not involved in fatty acid biosynthesis, but is primarily involved in stabilizing proteins involved in mitochondrial Fe-S complex formation. Analysis of mACP function in these protists indicates that ACP acquired a role in Fe-S complex formation early in eukaryotic evolution and highlights additional components of the Plasmodium respiratory chain that are important for viability.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Most eukaryotic cells retain a mitochondrial fatty acid synthesis (FASII) pathway whose acyl carrier protein (mACP) and 4-phosphopantetheine (Ppant) prosthetic group provide a soluble scaffold for acyl chain synthesis and biochemically couple FASII activity to mitochondrial electron transport chain (ETC) assembly and Fe-S cluster biogenesis. In contrast, the mitochondrion of Plasmodium falciparum malaria parasites lacks FASII enzymes yet curiously retains a divergent mACP lacking a Ppant group. We report that ligand-dependent knockdown of mACP is lethal to parasites, indicating an essential FASII-independent function. Decyl-ubiquinone rescues parasites temporarily from death, suggesting a dominant dysfunction of the mitochondrial ETC. Biochemical studies reveal that Plasmodium mACP binds and stabilizes the Isd11-Nfs1 complex required for Fe-S cluster biosynthesis, despite lacking the Ppant group required for this association in other eukaryotes, and knockdown of parasite mACP causes loss of Nfs1 and the Rieske Fe-S protein in ETC complex III. This work reveals that Plasmodium parasites have evolved to decouple mitochondrial Fe-S cluster biogenesis from FASII activity, and this adaptation is a shared metabolic feature of other apicomplexan pathogens, including Toxoplasma and Babesia . This discovery unveils an evolutionary driving force to retain interaction of mitochondrial Fe-S cluster biogenesis with ACP independent of its eponymous function in FASII.
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Author Response:
Reviewer #1:
The eukaryotic mitochondrial acyl carrier protein (mACP) has been shown to have two functions; as the acyl-chain carrier for FASII lipoic acid biosynthesis and as a chaperone for the heterodimeric cysteine desulphurase complex (IsD11-Nfs1) involved in the synthesis of Fe-S complexes. Previous studies have shown that the evolutionarily divergent protist, Plasmodium falciparum, lacks a mitochondrial FASII pathway but retains a putative mitochondrially located ACP. In this study, the P. falciparum mACP is shown to be essential for Fe-S complex formation, the assembly of the mitochondrial respiratory chain Complex III and the viability of red blood cell parasite stages. Using a conditional TetR knock-down system, pull-down experiments and homology modelling the authors demonstrate the mACP binds to the LYR …
Author Response:
Reviewer #1:
The eukaryotic mitochondrial acyl carrier protein (mACP) has been shown to have two functions; as the acyl-chain carrier for FASII lipoic acid biosynthesis and as a chaperone for the heterodimeric cysteine desulphurase complex (IsD11-Nfs1) involved in the synthesis of Fe-S complexes. Previous studies have shown that the evolutionarily divergent protist, Plasmodium falciparum, lacks a mitochondrial FASII pathway but retains a putative mitochondrially located ACP. In this study, the P. falciparum mACP is shown to be essential for Fe-S complex formation, the assembly of the mitochondrial respiratory chain Complex III and the viability of red blood cell parasite stages. Using a conditional TetR knock-down system, pull-down experiments and homology modelling the authors demonstrate the mACP binds to the LYR protein Isd11 via a novel interface and stabilizes Nfs1, which in turn is required for expression of the Rieske protein and Complex III function. The conclusions are well supported by the data which are of very high quality. This study is important in identifying new mitochondrial processes that are essential for Plasmodium infectivity. More broadly, the study highlights the important and evolutionarily conserved role that mACP has in assembly of Fe-S complexes in eukaryotic cells and the extent to which this function can be decoupled from FASII fatty acid biosynthesis.
We thank the reviewer for these positive comments.
Reviewer #2:
Major weaknesses:
Throughout the result and discussion the authors conclude that mACP is essential for Fe-S cluster biogenesis (importantly, lines 480-486, and figure 6, are extrapolating). However, while the interaction with Fe-S cluster biosynthesis pathway component is established, a role of mACP in Fe-S cluster biosynthesis or its control is implied from indirect evidence. It is possible that the depletion of complex III subunits and defect in mETC functions are an outcome of other mitochondrial defects. One example would be a defect in mitochondrial translation that leads to complex disassembly with an outcome on the abundance of nuclear encoded complex components.
We note that we formally show that mACP directly binds Isd11 and pulls down with Nfs1. We also show that loss of mACP is accompanied by loss of Nfs1, which is expected to ablate Fe-S cluster assembly. Consistent with this expectation, loss of mACP is accompanied by loss of the Rieske Fe-S protein. We agree with the reviewer that it remains an important future challenge to test the impact of mACP knockdown on other Fe-S proteins and thus more broadly study the impact of Nfs1 loss on other Fe-S client proteins and processes within and outside the mitochondrion. These studies are in progress.
Nsf1 and Rieske instability is used as support for defect in assembly of Fe-S cluster biosynthesis pathway and indirect support for defect in Fe-S cluster biogenesis. However, the data is not presented with independent repetitions and statistical analysis nor with quantification of the EF1 control. Moreover, it is not specified if the EF1 proteins used for control is mitochondrial. An unrelated mitochondrial protein that is not down-regulated is essential to support the conclusion about specific instability of NSf1 and Rieske.
We have included biological replicates of these experiments and used densitometry to quantify the levels of knockdown relative to loading controls. We have also added additional western blot analyses to Fig. 4 that show that levels of the mitochondrial chaperone Hsp60 and the ETC complex III protein, cyt c1, are unaffected by loss of mACP. The results strongly support our conclusion that mACP knockdown specifically reduces stability of Nfs1 and Rieske.
The characterisation of the mACP phenotype is driven by the elegant hypothesis that it performs the same alternative roles that other mACPs perform in addition to FASII in other organisms. However, the work ignores other possibilities - what is the effect of depletion on other mitochondrial functions (e.g. biogenesis pathways such as protein import, division and translation) and is the effect on mETC primary or secondary. Likewise, what is the effect on other cellular functions, is the mitochondrial defect primary?
The critical point here is that the essential interaction of mACP with Isd11-Nfs1 identified in our manuscript is sufficient to explain the observed phenotypes. We acknowledge that mACP may have other key interactions in parasites (that also contribute to mACP essentiality). However, any such interactions will be divergent relative to other studied eukaryotes and independent of known LYR-motif protein homologs that mediate conserved mACP functions in yeast and humans. As noted in our responses above, mACP knockdown does not result in mitochondrial depolarization and thus is not expected to inhibit import of mitochondrial- targeted proteins (which depends on the transmembrane potential), nor do observed phenotypes provide any evidence for impacts on mitochondrial translation.
A direct role is Fe-S cluster pathway assembly and Fe-S cluster biosynthesis is not directly established, and other mitochondrial functions are not examined. Finally, it is also not clear weather mitochondrial functions are the primary defect, since other cellular functions are not tested.
Please see our responses above. A full investigation of mitochondrial Fe-S cluster pathway assembly (which has not previously been studied in Plasmodium) is well beyond the scope of the present manuscript. We agree that future studies of broader Fe-S metabolism (beyond the scope of the present manuscript) will test and extend the conclusions of the present study. The new data added to Figure 4 strongly suggests that loss of mACP results specifically in loss of Nfs1 and Rieske and has no detectable impact on general mitochondrial proteins and functions that include retention of transmembrane potential, levels of the Hsp60 chaperone and the core complex III sub- unit cyt c1, and import of nuclear-encoded proteins into the mitochondrion and processing (e.g., Hsp60 and cyt c1) that require an intact transmembrane potential.
Reviewer #3:
Targeting of mACP to the parasite mitochondrion is nicely confirmed, as is essentiality via conditional knockdown. Some effort was undertaken to home in on leucine 51 as a likely point of cleavage for the removal of the mitochondrial targeting leader (Figure 1C & Figure 1 supplement 2). Is it possible to make a targeted search through the peptide hits and look for a peptide commencing with leucine 51 as a sort of poor man's N-terminome?
We thank the reviewer for this suggestion. We have on-going experiments to more precisely define the N-terminal processing site for mACP.
Binding of mACP to Isd11 is clearly demonstrated, as is further linking to Nsf1 to create a likely iron sulfur complex forming machine. I'm no structural biologist but it strikes me that a single protruding hydrophobic residue (F113) docked into a hydrophobic pocket on Isd11, plus a little cooperation from mACP V117, would make for a very weak interaction. Is that the sole binding interface? The mutagenesis (mACP F113A) abrogating pull down by nickel chromatography when expressed heterologously in bacteria is compelling. Are there data to show this pull down fails in the presence of detergent? Are there comparable examples of weak hydrophobic interactions generating such good binding?
To clarify, the binding interface of the Plasmodium Isd11-mACP complex spans many intermolecular interactions beyond the predicted role for F113. This predicted interface includes electrostatic interactions between conserved Asp/Glu residues on mACP and conserved Lys/Arg residues on Isd11. The residues involved in these predicted intermolecular electrostatic interactions appear to be conserved between the parasite proteins and Isd11 and mACP from yeast to humans.
The key molecular difference in the parasite binding interface is due to specific loss of a negatively charged phosphopantetheine group on mACP and its replacement with a hydrophobic F. In parallel, the parasite Isd11 R6I modification has replaced the positively charged group that canonically interacts with the phosphopanthetheine oxyanions with a hydrophobic isoleucine. These adaptive interactions change the local environment of the mACP-Isd11 binding interface near mACP residue 113 to one that is more hydrophobic. Our F113A mutagenesis results highlight the important contribution of F113 to the stability of this interface, even though the interface spans a much larger molecular surface with additional electrostatic interactions. We have on-going structural and biochemical studies to fully understand the intermolecular interactions that stabilize association of Isd11 and mACP in Plasmodium.
We have modified the text to clarify that additional conserved electrostatic interactions also contribute to stability of the mACP-Isd11 interface. We have also added Figure 3- figure supplement 1 to explicitly show these electrostatic interactions in the parasite complex and now include the PDB file of the Rosetta model as source data so readers can view the low-energy model themselves.
Line 900 - Figure 2 supplements 4 & 5. There appear to be many spectral counts in the table of mass spec hits for mAPC/Isd11 complex retrieved from bacteria by nickel chromatography, but only one peptide (being the largest possible) is displayed in supplement 5. Is there a reason that smaller, sub-peptides were not observed?
To clarify, Figure 2- figure supplement 4 (figure supplement 5 in the revised manuscript) is data from the IP/MS analysis of parasites expressing mACP-HA2 or aACP-HA2 that establishes similar detection of both bait proteins despite only substantial detection of Nfs1 in the IP sample for mACP-HA2. Figure 2- figure supplement 5 (figure supplement 6 in the revised manuscript) is data based on recombinant expression of Isd11 and mACP in E. coli. For IP/MS-MS of the recombinant proteins, we did indeed observe smaller tryptic peptides. The underlined region was simply meant to convey the overall sequence coverage spanned by the observed peptides. We have modified the legend of the revised Figure 2- figure supplement 6 to clarify the overall sequence coverage from IP/MS-MS analysis and now include lines for individual peptides.
On line 548 the authors speculate that a small molecule inhibitor of the protein/protein interaction between mACP and Isd11 might be a pan-apicomplexan drug. To better substantiate this speculation, it would be nice to include an alignment of the chromerid and apicomplexan mACP proteins to illustrate the apparent switch from a 4-phosphopantetheine prosthetic group attached via serine to a phenylalanine and the postulated hydrophobic binding interaction.
We have followed the reviewer’s suggestion and now include this alignment as Figure 6- figure supplement 1.
Why was 1µm proguanil used for the 7 day growth assay (Fig 5A) and 5 day assay (Fig 5B) but 5µm for the MitoTracker imaging?
For growth assays spanning several days, we used the lower proguanil concentration to avoid any toxicity from proguanil alone, which can inhibit parasite growth at IC50 ~10 μM via targets that are poorly defined but appear to be independent of the parasite electron transport chain (discussed in ref. 46). Because the imaging experiment in Figure 5C involved only two days of proguanil treatment and was primarily concerned with impacts on MitoTracker staining rather than parasite growth, we used a higher 5 μM proguanil concentration. This proguanil concentration by itself had no impact on MitoTracker accumulation in the mitochondrion but resulted in dispersed signal when combined with mACP knockdown -aTc.
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Evaluation Summary:
This study defines the role of a divergent mitochondrial-localized isoform of a FASII acyl carrier protein (mACP) in the malaria parasite, Plasmodium falciparum. In contrast to the situation in other eukaryotes, mACP is not involved in fatty acid biosynthesis, but is primarily involved in stabilizing proteins involved in mitochondrial Fe-S complex formation. Analysis of mACP function in these protists indicates that ACP acquired a role in Fe-S complex formation early in eukaryotic evolution and highlights additional components of the Plasmodium respiratory chain that are important for viability.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 …
Evaluation Summary:
This study defines the role of a divergent mitochondrial-localized isoform of a FASII acyl carrier protein (mACP) in the malaria parasite, Plasmodium falciparum. In contrast to the situation in other eukaryotes, mACP is not involved in fatty acid biosynthesis, but is primarily involved in stabilizing proteins involved in mitochondrial Fe-S complex formation. Analysis of mACP function in these protists indicates that ACP acquired a role in Fe-S complex formation early in eukaryotic evolution and highlights additional components of the Plasmodium respiratory chain that are important for viability.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The eukaryotic mitochondrial acyl carrier protein (mACP) has been shown to have two functions; as the acyl-chain carrier for FASII lipoic acid biosynthesis and as a chaperone for the heterodimeric cysteine desulphurase complex (IsD11-Nfs1) involved in the synthesis of Fe-S complexes. Previous studies have shown that the evolutionarily divergent protist, Plasmodium falciparum, lacks a mitochondrial FASII pathway but retains a putative mitochondrially located ACP. In this study, the P. falciparum mACP is shown to be essential for Fe-S complex formation, the assembly of the mitochondrial respiratory chain Complex III and the viability of red blood cell parasite stages. Using a conditional TetR knock-down system, pull-down experiments and homology modelling the authors demonstrate the mACP binds to the LYR …
Reviewer #1 (Public Review):
The eukaryotic mitochondrial acyl carrier protein (mACP) has been shown to have two functions; as the acyl-chain carrier for FASII lipoic acid biosynthesis and as a chaperone for the heterodimeric cysteine desulphurase complex (IsD11-Nfs1) involved in the synthesis of Fe-S complexes. Previous studies have shown that the evolutionarily divergent protist, Plasmodium falciparum, lacks a mitochondrial FASII pathway but retains a putative mitochondrially located ACP. In this study, the P. falciparum mACP is shown to be essential for Fe-S complex formation, the assembly of the mitochondrial respiratory chain Complex III and the viability of red blood cell parasite stages. Using a conditional TetR knock-down system, pull-down experiments and homology modelling the authors demonstrate the mACP binds to the LYR protein Isd11 via a novel interface and stabilizes Nfs1, which in turn is required for expression of the Rieske protein and Complex III function. The conclusions are well supported by the data which are of very high quality. This study is important in identifying new mitochondrial processes that are essential for Plasmodium infectivity. More broadly, the study highlights the important and evolutionarily conserved role that mACP has in assembly of Fe-S complexes in eukaryotic cells and the extent to which this function can be decoupled from FASII fatty acid biosynthesis.
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Reviewer #2 (Public Review):
A summary of what the authors aimed to achieve:
Seeing that Plasmodium's FAS II pathway is hosted in the apicoplast, the role of a putative mitochondrial ACP is an enigma. The curious nature of this puzzle is enhanced by evidence that mACP is essential in red blood stages where few of the mitochondrial metabolic pathways are essential. The authors set to examine if mACP may have alternative functions to FASII, such as respiratory chain complex assembly, Fe-S biosynthesis and translation, as shown for mACP in other eukaryotes in addition to a role if FAS II.
Furthermore, the Plasmodium mACP lack the 4-phosphopantetheine (Ppant) prosthetic group, whereas the acylation of mACP of other organisms is necessary for these "alternative" functions, because it mediates interactions with the so called LYR-proteins that …
Reviewer #2 (Public Review):
A summary of what the authors aimed to achieve:
Seeing that Plasmodium's FAS II pathway is hosted in the apicoplast, the role of a putative mitochondrial ACP is an enigma. The curious nature of this puzzle is enhanced by evidence that mACP is essential in red blood stages where few of the mitochondrial metabolic pathways are essential. The authors set to examine if mACP may have alternative functions to FASII, such as respiratory chain complex assembly, Fe-S biosynthesis and translation, as shown for mACP in other eukaryotes in addition to a role if FAS II.
Furthermore, the Plasmodium mACP lack the 4-phosphopantetheine (Ppant) prosthetic group, whereas the acylation of mACP of other organisms is necessary for these "alternative" functions, because it mediates interactions with the so called LYR-proteins that are involved in those functions. Thus, the authors explored weather an interaction with LYR proteins occurs in Plasmodium, and how it is mediated in the absence of Ppant.
An account of the major strengths and weaknesses of the methods and results:
Major strengths:
Since mATC wasn't studied in Plasmodium, it was necessary to confirm its mitochondrial localisation and assess its essential role in blood stages, for which the authors provided completing evidence.
In light of the lack of FASII in Plasmodium mitochondria and known role for mACP in other mitochondrial function via interaction with LYR proteins, the authors identify a Plasmodium LYR protein to be homolog of the Fe-S biosyntheis component Isd11 and demonstrate direct interaction between PfmACP and Isd11 via reciprocal co-IP of tagged proteins, and through a heterologous system. Unbiased IP and MS further identify Nsf1, provides additional support to interaction with the Fe-S synthesis pathway.
Through sequence analysis and comparison to the described interaction between mACP and Isd11 in other systems, the authors formulate an elegant hypothesis about their co-evolution in Plasmodium. This is backed with nice studied of the amino acid required for their interaction.
Finally, the authors provide evidence for mACP essentiality in blood staged and deliver extensive characterisation of the defect of mETC function in the mitochondrial of parasites depleted from mACP.
Major weaknesses:
Throughout the result and discussion the authors conclude that mACP is essential for Fe-S cluster biogenesis (importantly, lines 480-486, and figure 6, are extrapolating). However, while the interaction with Fe-S cluster biosynthesis pathway component is established, a role of mACP in Fe-S cluster biosynthesis or its control is implied from indirect evidence. It is possible that the depletion of complex III subunits and defect in mETC functions are an outcome of other mitochondrial defects. One example would be a defect in mitochondrial translation that leads to complex disassembly with an outcome on the abundance of nuclear encoded complex components.
Nsf1 and Rieske instability is used as support for defect in assembly of Fe-S cluster biosynthesis pathway and indirect support for defect in Fe-S cluster biogenesis. However, the data is not presented with independent repetitions and statistical analysis nor with quantification of the EF1 control. Moreover, it is not specified if the EF1 proteins used for control is mitochondrial. An unrelated mitochondrial protein that is not down-regulated is essential to support the conclusion about specific instability of NSf1 and Rieske.
The characterisation of the mACP phenotype is driven by the elegant hypothesis that it performs the same alternative roles that other mACPs perform in addition to FASII in other organisms. However, the work ignores other possibilities - what is the effect of depletion on other mitochondrial functions (e.g. biogenesis pathways such as protein import, division and translation) and is the effect on mETC primary or secondary. Likewise, what is the effect on other cellular functions, is the mitochondrial defect primary?
An appraisal of whether the authors achieved their aims, and whether the results support their conclusions:
The authors establish that mACP is mitochondrial, and that it binds the LYR protein they had identified. They further established the mACP is essential for blood stages and that its depletion results in defects in mitochondrial mETC function. Finally, they provide a nice characterisation of the potential changes in interaction between mACP and a LYR protein that compensate for the lack of Ppant.
A direct role is Fe-S cluster pathway assembly and Fe-S cluster biosynthesis is not directly established, and other mitochondrial functions are not examined. Finally, it is also not clear weather mitochondrial functions are the primary defect, since other cellular functions are not tested.
A discussion of the likely impact of the work on the field, and the utility of the methods and data to the community:
The work will have impact on the understanding of cellular evolution as it highlights similarities and differences to the functions and mechanism of partner interactions of mACT between a model organism of the apicomplexans and previous observations from human and yeast.
The work also unravels a new essential function in the disease-causing stage of Plasmodium. This has potential to produce impact via informing drug discovery efforts with a putative new target, however will be improved by further characterisation of mACP functions as mentioned in the weaknesses.
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Reviewer #3 (Public Review):
The authors set out to explore the function of mitochondrial acyl carrier protein (mACP) in the malaria parasite Plasmodium falciparum given that no acyl chains are 'carried' in the parasite mitochondrion because it does not make acyl chains therein.
Targeting of mACP to the parasite mitochondrion is nicely confirmed, as is essentiality via conditional knockdown. Some effort was undertaken to home in on leucine 51 as a likely point of cleavage for the removal of the mitochondrial targeting leader (Figure 1C & Figure 1 supplement 2). Is it possible to make a targeted search through the peptide hits and look for a peptide commencing with leucine 51 as a sort of poor man's N-terminome?
Binding of mACP to Isd11 is clearly demonstrated, as is further linking to Nsf1 to create a likely iron sulfur complex forming …
Reviewer #3 (Public Review):
The authors set out to explore the function of mitochondrial acyl carrier protein (mACP) in the malaria parasite Plasmodium falciparum given that no acyl chains are 'carried' in the parasite mitochondrion because it does not make acyl chains therein.
Targeting of mACP to the parasite mitochondrion is nicely confirmed, as is essentiality via conditional knockdown. Some effort was undertaken to home in on leucine 51 as a likely point of cleavage for the removal of the mitochondrial targeting leader (Figure 1C & Figure 1 supplement 2). Is it possible to make a targeted search through the peptide hits and look for a peptide commencing with leucine 51 as a sort of poor man's N-terminome?
Binding of mACP to Isd11 is clearly demonstrated, as is further linking to Nsf1 to create a likely iron sulfur complex forming machine. I'm no structural biologist but it strikes me that a single protruding hydrophobic residue (F113) docked into a hydrophobic pocket on Isd11, plus a little cooperation from mACP V117, would make for a very weak interaction. Is that the sole binding interface? The mutagenesis (mACP F113A) abrogating pull down by nickel chromatography when expressed heterologously in bacteria is compelling. Are there data to show this pull down fails in the presence of detergent? Are there comparable examples of weak hydrophobic interactions generating such good binding?
Line 900 - Figure 2 supplements 4 & 5. There appear to be many spectral counts in the table of mass spec hits for mAPC/Isd11 complex retrieved from bacteria by nickel chromatography, but only one peptide (being the largest possible) is displayed in supplement 5. Is there a reason that smaller, sub-peptides were not observed?
On line 548 the authors speculate that a small molecule inhibitor of the protein/protein interaction between mACP and Isd11 might be a pan-apicomplexan drug. To better substantiate this speculation, it would be nice to include an alignment of the chromerid and apicomplexan mACP proteins to illustrate the apparent switch from a 4-phosphopantetheine prosthetic group attached via serine to a phenylalanine and the postulated hydrophobic binding interaction.
Why was 1µm proguanil used for the 7 day growth assay (Fig 5A) and 5 day assay (Fig 5B) but 5µm for the MitoTracker imaging?
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