A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites

Tang, Yong; Meister, Thomas R.; Walczak, Marta; Pulkoski-Gross, Michael J.; Hari, Sanjay B.; Sauer, Robert T.; Amberg-Johnson, Katherine; Yeh, Ellen

doi:10.1101/401570

Cited by 8 publications

(16 citation statements)

References 69 publications

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“…We previously performed a mutagenesis screen to identify new apicoplast biogenesis genes ( 14 ). Loss-of-function mutations in these genes disrupt inheritance of the apicoplast during parasite replication, resulting in organelle loss in daughter cells.…”

Section: Resultsmentioning

confidence: 99%

“…As a result of its divergent evolution, thousands of genes in the P. falciparum genome remain unannotated ( 38 ). We previously designed an unbiased screen that allows for discovery of new genes with essential roles in apicoplast biogenesis ( 14 ). Here, we validate a previously uncharacterized gene candidate from our screen, AMR4, and show that it has an essential function in apicoplast biogenesis.…”

Section: Discussionmentioning

confidence: 99%

“…Isolation of apicoplast-minus mutant clones and DNA extraction were described previously ( 14 ). Genomic DNA samples from six additional clones were used to generate paired-end libraries using the NEB Next Ultra FS II kit with eight PCR cycles.…”

Section: Methodsmentioning

confidence: 99%

“…The transit of nucleus-encoded proteins to the apicoplast requires vesicle trafficking from the endoplasmic reticulum (ER) and a unique s ymbiont-derived E R-associated degradation- l ike ma chinery (SELMA) to cross the two outer membranes ( 7 – 10 ). Additionally, several members of the highly conserved eukaryotic autophagy pathway are unexpectedly required for apicoplast inheritance ( 11 – 14 ). While the repurposed SELMA and autophagy components hint at an abundance of molecular innovation in apicoplast biogenesis pathways, most of these pathways remain undiscovered.…”

Section: Introductionmentioning

confidence: 99%

“…To discover new genes required for apicoplast biogenesis and uncover more instances of molecular innovation, we previously conducted a mutagenesis screen to identify mutations that cause apicoplast loss ( 14 ). From this screen, we identified a mutation in a gene encoding a CaaX protease and bacteriocin processing (CPBP) family protein (Pf3D7_0913500, S347G).…”

Section: Introductionmentioning

confidence: 99%

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CaaX-Like Protease of Cyanobacterial Origin Is Required for Complex Plastid Biogenesis in Malaria Parasites

Meister

Tang

Pulkoski-Gross

et al. 2020

mBio

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Plasmodium parasites and related apicomplexans contain an essential “complex plastid” organelle of secondary endosymbiotic origin, the apicoplast. Biogenesis of this complex plastid poses a unique challenge requiring evolution of new cellular machinery. We previously conducted a mutagenesis screen for essential apicoplast biogenesis genes to discover organellar pathways with evolutionary and biomedical significance. Here we validate and characterize a gene candidate from our screen, Pf3D7_0913500. Using a conditional knockdown strain, we show that Pf3D7_0913500 depletion causes growth inhibition that is rescued by the sole essential product of the apicoplast, isopentenyl pyrophosphate (IPP), and results in apicoplast loss. Because Pf3D7_0913500 had no previous functional annotation, we name it apicoplast-minus IPP-rescued 4 (AMR4). AMR4 has an annotated CaaX protease and bacteriocin processing (CPBP) domain, which in eukaryotes typically indicates a role in CaaX postprenylation processing. Indeed, AMR4 is the only putative CaaX-like protease in Plasmodium parasites which are known to require protein prenylation, and we confirm that the conserved catalytic residue of AMR4 (E352) is required for its apicoplast function. However, we unexpectedly find that AMR4 does not act in a CaaX postprenylation processing pathway in Plasmodium falciparum. Instead, we find that AMR4 is imported into the apicoplast and is derived from a cyanobacterial CPBP gene which was retained through both primary and secondary endosymbiosis. Our findings suggest that AMR4 is not a true CaaX protease, but instead it performs a conserved, uncharacterized chloroplast function that has been retained for complex plastid biogenesis. IMPORTANCE Plasmodium parasites, which cause malaria, and related apicomplexans are important human and veterinary pathogens. These parasites represent a highly divergent and understudied branch of eukaryotes, and as such often defy the expectations set by model organisms. One striking example of unique apicomplexan biology is the apicoplast, an essential but nonphotosynthetic plastid derived from an unusual secondary (eukaryote-eukaryote) endosymbiosis. Endosymbioses are a major driver of cellular innovation, and apicoplast biogenesis pathways represent a hot spot for molecular evolution. We previously conducted an unbiased screen for apicoplast biogenesis genes in P. falciparum to uncover these essential and innovative pathways. Here, we validate a novel gene candidate from our screen and show that its role in apicoplast biogenesis does not match its functional annotation predicted by model eukaryotes. Our findings suggest that an uncharacterized chloroplast maintenance pathway has been reused for complex plastid biogenesis in this divergent branch of pathogens.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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CaaX-Like Protease of Cyanobacterial Origin Is Required for Complex Plastid Biogenesis in Malaria Parasites

Meister

Tang

Pulkoski-Gross

et al. 2020

mBio

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Essential Genes of the Parasitic Apicomplexa

Oberstaller

Otto

Rayner

et al. 2021

Trends in Parasitology

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Identification and phylogenetic analysis of RNA binding domain abundant in apicomplexans or RAP proteins

et al. 2021

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The RNA binding domain abundant in apicomplexans (RAP) is a protein domain identified in a diverse group of proteins, called RAP proteins, many of which have been shown to be involved in RNA binding. To understand the expansion and potential function of the RAP proteins, we conducted a hidden Markov model based screen among the proteomes of 54 eukaryotes, 17 bacteria and 12 archaea. We demonstrated that the domain is present in closely and distantly related organisms with particular expansions in Alveolata and Chlorophyta, and are not unique to Apicomplexa as previously believed. All RAP proteins identified can be decomposed into two parts. In the N-terminal region, the presence of variable helical repeats seems to participate in the specific targeting of diverse RNAs, while the RAP domain is mostly identified in the C-terminal region and is highly conserved across the different phylogenetic groups studied. Several conserved residues defining the signature motif could be crucial to ensure the function(s) of the RAP proteins. Modelling of RAP domains in apicomplexan parasites confirmed an ⍺/β structure of a restriction endonuclease-like fold. The phylogenetic trees generated from multiple alignment of RAP domains and full-length proteins from various distantly related eukaryotes indicated a complex evolutionary history of this family. We further discuss these results to assess the potential function of this protein family in apicomplexan parasites.

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A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites

Cited by 8 publications

References 69 publications

CaaX-Like Protease of Cyanobacterial Origin Is Required for Complex Plastid Biogenesis in Malaria Parasites

CaaX-Like Protease of Cyanobacterial Origin Is Required for Complex Plastid Biogenesis in Malaria Parasites

Essential Genes of the Parasitic Apicomplexa

Identification and phylogenetic analysis of RNA binding domain abundant in apicomplexans or RAP proteins

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