Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of NTH

Protein (function)
Genome analysis shows that Plasmodium species encode a single, conserved membrane-bound NAD(P) transhydrogenases (NTH).
Plasmodium berghei NTH is encoded by a three-exon gene and is composed of 1,201 amino acids with a calculated Mr of 135,198 and has a predicted amino-terminal ER signal peptide sequence that forms part of a bipartite apicoplast targeting sequence.  The gene product forms 11 predicted transmembrane helices and has additional domains for binding NAD(H) and NADP(H). This structure conforms with the general architecture of proton-translocating NTH proteins, consisting of three functional domains: domain I, which binds NAD(H); domain II, which contains the membrane-spanning helices, and domain III, which binds NADP(H). Functional domains I and III together facilitate hydride transfer between NAD(H) and NADP (H), whereas domain II facilitates proton translocation across the lipid bilayer in which the NTH protein is embedded. Whilst prokaryotes have segmented nth genes, eukaryotic nth genes are unsegmented and encode single polypeptide NTH proteins, either linking the a subunit C-terminus to the β subunit N-terminus (αβ) as illustrated by mammalian NTH or linking the b subunit C-terminus to the a subunit N-terminus (ba) as illustrated by Plasmodium NTH. Accordingly, the order of domains in Plasmodium NTH is dIIa-dIII-dI-dIIb.
NAD(P) transhydrogenases (NTHs) are enzymes that catalyse the reversible hydride ion transfer between NAD(H) and NADP(H). They exist as soluble (EC 1.6.1.1) and membrane-bound (EC 1.6.1.2) isoforms. The latter are integral multi-pass membrane proteins that in bacteria reside in the cytoplasmic membrane, whilst in metazoans they are situated in the inner membrane of mitochondria, likely reflecting the evolutionary origin of the mitochondrion from a bacterial primary endosymbiont The widely accepted view of the physiological role of mitochondrial NTH is to generate NADPH (the reduced form of NADP), either required for mitochondrion-specific biosynthetic purposes, or to protect the organelle from oxidative damage caused by free radicals generated in the respiratory chain.
In this study, we characterise a membrane-bound NTH in malaria parasites that is not present in mitochondria, but instead localises in the crystalloid, an enigmatic organelle found in ookinetes and young oocysts that is critically involved in sporogony. We show that NTH has an essential structural role in crystalloid formation, as well as a vital enzymatic role in sporogony, indicating that the organelle requires NADPH to function. NTH is also found in the sporozoite apicoplast, addressing a longstanding question about the potential source of NADPH required for some of the anabolic activities that take place in this plastid of likely red algal origin.

Phenotype
Analyses of a mutant lacking expression of NTH showed (RMgm-4727):
Normal fertilisation and normal numbers of ookinetes produced. However, pigment in these ookinetes was more dispersed and not found in clusters that typically surround and highlight the crystalloids. Normal numbers of ookinetes are produced. However, despite undergoing substantial nuclear expansion these oocysts failed to produce sporozoites.
These collective findings demonstrate that NTH is required for crystalloid biogenesis and sporozoite development.

Analyses of the mutant expressing a C-terminal GFP tagged version of NTH showed:
The resulting parasites (termed NTH/GFP) developed normally in mouse and mosquito and were transmitted by mosquito bite, indicating that the GFP tag had not interfered with NTH function. In the mouse, neither asexual nor sexual blood-stage parasites displayed discernible GFP-based fluorescence. Dispersed extranuclear GFP fluorescence was first detected in zygotes ~ 4 h after gametocyte activation consistent with a post-fertilisation lifting of translational repression and a localisation of the protein in the ER. By 24 h, mature ookinetes showed GFP fluorescence that was concentrated in typically two spots associated with pigment clusters. This distribution pattern is  characteristic of proteins that are trafficked to the crystalloids. Previous studies have shown that a protein complex of six modular proteins rich in putative carbohydrate binding domains, named LCCL lectin adhesive-like proteins (LAPs), resides in the crystalloid. To confirm the localisation of NTH in the crystalloid, we co-localised NTH with LAP3. To achieve this, a parasite line expressing mCherry-tagged LAP3 (named LAP3/mCherry; see RMgm-1473)) was genetically crossed in vitro with parasite line NTH/GFP. Subsequent infection of mosquitoes with the resultant ookinete population gives rise to heterokaryotic polyploid oocysts containing mixtures of parental, wild-type, and double gene-tagged (i.e. possessing modified alleles for both nth and lap3) sporozoites. The ensuing sporozoite population was transmitted to naive mice by mosquito bite and drug selected. Ookinete cultures derived from the transmitted parasite population contained zygotes and ookinetes with dual expression of NTH::GFP and LAP3::mCherry that co-localised both before and after crystalloid formation, respectively.

Apart from very young oocysts that still possess crystalloids, no discernible GFP fluorescence was observed in oocysts before sporulation. However, GFP fluorescence was observed in sporulated oocysts and in individual sporozoites, indicating that NTH is once again expressed in sporozoites. Sporozoite NTH was concentrated in a tubular structure reminiscent of the apicoplast, consistent with NTH possessing a predicted apicoplast transit peptide. To confirm apicoplast targeting of NTH, we co-localised it with the apicoplastresident acyl carrier protein (ACP). To achieve this, ACP was fused to the red fluorescent protein mCherry and the resulting parasite line (named ACP/mCherry) was genetically crossed with parasite line NTH/GFP. As ACP::mCherry was driven from a sporozoite-specific promoter, resultant heterokaryotic polyploid oocysts produced sporozoites that simultaneously expressed NTH::GFP and ACP::mCherry that localised to the same tubular structure. Co-localisation of green and red fluorescence in the organelle was partial, pointing to their presence in distinct apicoplast compartments and possibly reflecting the predicted localisation of NTH and ACP in membrane and stroma, respectively. Finally, co-staining of NTH/GFP sporozoites with MitoTracker Red confirmed that NTH was absent from the mitochondrion. The clear spatial separation of apicoplast and mitochondrion confirms previous observations that these two organelles are not physically connected in sporozoites. In contrast to crystalloids, the apicoplast is present in the parasite throughout the life cycle, and nuclear-encoded apicoplast proteins are delivered in vesicles to a ready-formed organelle. Protein trafficking to the apicoplast also occurs via the ER and involves a bipartite amino-terminal signal composed of an amino-terminal ER signal peptide followed by an apicoplast transit peptide, both of which are present in NTH. Although the sporozoite-specific expression of ACP::mCherry in our ACP/mCherry line precluded assessment of co-localisation with NTH::GFP in zygotes/ookinetes by genetic crossing, GFP fluorescence alone did not show evidence for a localisation of NTH in the apicoplast of these life stages. This indicates that in these life stages crystalloid targeting overrides apicoplast targeting. One explanation is that NTH is delivered to the crystalloid from the ER already in complex with the LAPs and/or other crystalloid proteins, preventing its trafficking to the apicoplast in the same cell. Because crystalloids are not present in sporozoites, the apicoplast becomes the default target organelle for NTH in this life cycle stage.

Additional information
Various Plasmodium transcriptome studies identified transcripts of the nth gene predominantly in female gametocytes and to be translationally repressed. T

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