The fragmented mitochondrial ribosomal RNAs of Plasmodium falciparum.

BACKGROUND: The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. PRINCIPAL FINDINGS: The identification of 14 additional small mitochondrial transcripts from P. falciparum and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. SIGNIFICANCE: All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered.

Branch point identification and sequence requirements for intron splicing in Plasmodium falciparum.

Splicing of mRNA is an ancient and evolutionarily conserved process in eukaryotic organisms, but intron-exon structures vary. Plasmodium falciparum has an extreme AT nucleotide bias (>80%), providing a unique opportunity to investigate how evolutionary forces have acted on intron structures. In this study, we developed an in vivo luciferase reporter splicing assay and employed it in combination with lariat isolation and sequencing to characterize 5' and 3' splicing requirements and experimentally determine the intron branch point in P. falciparum. This analysis indicates that P. falciparum mRNAs have canonical 5' and 3' splice sites. However, the 5' consensus motif is weakly conserved and tolerates nucleotide substitution, including the fifth nucleotide in the intron, which is more typically a G nucleotide in most eukaryotes. In comparison, the 3' splice site has a strong eukaryotic consensus sequence and adjacent polypyrimidine tract. In four different P. falciparum pre-mRNAs, multiple branch points per intron were detected, with some at U instead of the typical A residue. A weak branch point consensus was detected among 18 identified branch points. This analysis indicates that P. falciparum retains many consensus eukaryotic splice site features, despite having an extreme codon bias, and possesses flexibility in branch point nucleophilic attack.

Sequential processing of the Toxoplasma apicoplast membrane protein FtsH1 in topologically distinct domains during intracellular trafficking.

FtsH proteins are hexameric transmembrane proteases found in chloroplasts, mitochondria and bacteria. In the protozoan Toxoplasma gondii, FtsH1 is localized to membranes of the apicoplast, a relict chloroplast present in many apicomplexan parasites. We have shown that although T. gondii FtsH1 lacks the typical bipartite targeting presequence seen on apicoplast luminal proteins, it is targeted to the apicoplast via the endoplasmic reticulum. In this report, we show that FtsH1 undergoes processing events to remove both the N- and C-termini, which are topologically separated by the membrane in which FtsH1 is embedded. Pulse-chase analysis showed that N-terminal cleavage precedes C-terminal cleavage. Unlike the processing of the N-terminal transit peptide of luminal proteins, which occurs in the apicoplast, analysis of ER-retained mutants showed that N-terminal processing of FtsH1 occurs in the endoplasmic reticulum. Two of four FtsH1 mutants bearing internal epitope tags accumulated in structures peripheral to the apicoplast, implying that FtsH1 trafficking is highly sensitive to changes in protein structure. These mutant proteins did not undergo C-terminal processing, suggesting that this processing step occurs after localization to the plastid. Mutation of the peptidase active site demonstrated that neither processing event occurs in cis. These data support a model in which multiple proteases act at different points of the trafficking pathway to form mature FtsH1, making its processing more complex than other FtsHs and unique among apicoplast proteins described thus far.

A thioredoxin family protein of the apicoplast periphery identifies abundant candidate transport vesicles in Toxoplasma gondii.

Toxoplasma gondii, which causes toxoplasmic encephalitis and birth defects, contains an essential chloroplast-related organelle to which proteins are trafficked via the secretory system. This organelle, the apicoplast, is bounded by multiple membranes. In this report we identify a novel apicoplast-associated thioredoxin family protein, ATrx1, which is predominantly soluble or peripherally associated with membranes, and which localizes primarily to the outer compartments of the organelle. As such, it represents the first protein to be identified as residing in the apicoplast intermembrane spaces. ATrx1 lacks the apicoplast targeting sequences typical of luminal proteins. However, sequences near the N terminus are required for proper targeting of ATrx1, which is proteolytically processed from a larger precursor to multiple smaller forms. This protein reveals a population of vesicles, hitherto unrecognized as being highly abundant in the cell, which may serve to transport proteins to the apicoplast.

A membrane protease is targeted to the relict plastid of toxoplasma via an internal signal sequence.

The apicoplast is a secondary plastid found in Toxoplasma gondii, Plasmodium species and many other apicomplexan parasites. Although the apicoplast is essential to parasite survival, little is known about the protein constituents of the four membranes surrounding the organelle. Luminal proteins are directed to the endoplasmic reticulum (ER) by an N-terminal signal sequence and from there to the apicoplast by a transit peptide domain. We have identified a membrane-associated AAA protease in T. gondii, FtsH1. Although the protein lacks a canonical bipartite-targeting sequence, epitope-tagged FtsH1 colocalizes with the recently identified apicoplast membrane marker APT1 and immunoelectron microscopy confirms the residence of FtsH1 on plastid membranes. Trafficking appears to occur via the ER because deletion mutants lacking the peptidase domain are retained in the ER. When extended to include the peptidase domain, the protein trafficks properly. The transmembrane domain is required for localization of the full-length protein to the apicoplast and a truncation mutant to the ER. Thus, at least two distinct regions of FtsH1 are required for proper trafficking, but they differ from those of luminal proteins and would not be detected by the algorithms currently used to identify apicoplast proteins.

Cell cycle-regulated vesicular trafficking of Toxoplasma APT1, a protein localized to multiple apicoplast membranes.

The apicoplast is a relict plastid essential for viability of the apicomplexan parasites Toxoplasma and Plasmodium. It is surrounded by multiple membranes that proteins, substrates and metabolites must traverse. Little is known about apicoplast membrane proteins, much less their sorting mechanisms. We have identified two sets of apicomplexan proteins that are homologous to plastid membrane proteins that transport phosphosugars or their derivatives. Members of the first set bear N-terminal extensions similar to those that target proteins to the apicoplast lumen. While Toxoplasma gondii lacks this type of translocator, the N-terminal extension from the Plasmodium falciparum sequence was shown to be functional in T. gondii. The second set of translocators lacks an N-terminal targeting sequence. This translocator, TgAPT1, when tagged with HA, localized to multiple apicoplast membranes in T. gondii. Contrasting with the constitutive targeting of luminal proteins, the localization of the translocator varied during the cell cycle. Early-stage parasites showed circumplastid distribution, but as the plastid elongated in preparation for division, vesicles bearing TgAPT1 appeared adjacent to the plastid. After plastid division, the protein resumes a circumplastid colocalization. These studies demonstrate for the first time that vesicular trafficking likely plays a role in the apicoplast biogenesis.

Divergent polyamine metabolism in the Apicomplexa.

The lead enzymes of polyamine biosynthesis, i.e. ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), were not detected in Toxoplasma gondii [the limit of detection for ODC and ADC was 5 pmol min(-1) (mg protein)(-1)], indicating that T. gondii lacks a forward-directed polyamine biosynthetic pathway, and is therefore a polyamine auxotroph. The biochemical results were supported by results obtained from data-mining the T. gondii genome. However, it was possible to demonstrate the presence of a highly active backconversion pathway that formed spermidine from spermine, and putrescine from spermidine, via the combined action of spermidine/spermine N(1)-acetyltransferase (SSAT) or spermidine N(1)-acetyltransferase (SAT) and polyamine oxidase (PAO). With spermine as the substrate, T. gondii SSAT had a specific activity of 1.84 nmol min(-1) (mg protein)(-1), and an apparent K(m) for spermine of 180 mM; with spermidine as the substrate, the SAT had a specific activity of 3.95 nmol min(-1) (mg protein)(-1), and a K(m) for spermidine of 240 mM. T. gondii PAO had a specific activity of 10.6 nmol min(-1) (mg protein)(-1), and a K(m) for acetylspermine of 36 mM. Furthermore, the results demonstrated that T. gondii SSAT was 50 % inhibited by 30 mM di(ethyl)norspermine. The parasite actively transported arginine and ornithine, which were converted via the arginine dihydrolase pathway to citrulline and carbamoyl phosphate, resulting in the formation of ATP via carbamate kinase. The lack of polyamine biosynthesis by T. gondii is contrasted with polyamine metabolism by other apicomplexans.

Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting.

The apicoplast is a relict plastid found in many apicomplexans, including the pathogens Toxoplasma gondii and Plasmodium falciparum. Nucleus-encoded apicoplast proteins enter the ER, and after cleavage of the signal sequence, are routed to the apicoplast by virtue of a transit peptide, which is subsequently removed. To assess the mechanisms of localization we examined stable transfectants of T. gondii for the localization and processing of various GFP fusion proteins. GFP fusions bearing apicoplast targeting sequences targeted efficiently to the plastid, with no retention in the ER, even when an ER retention/retrieval sequence was added. Incubation with brefeldin A, which blocks ER-to-Golgi trafficking by inhibiting a GTP exchange factor required for retrograde trafficking, blocked the processing of the protein. Surprisingly, it did not affect the immunofluorescence pattern. To avoid the potentially misleading presence of pre-existing GFP fusion protein in the apicoplast, we used a ligand-regulated aggregation system to arrest the GFP fusion protein in the ER prior to trafficking. Upon addition of ligand to promote disaggregation, the fusion protein targeted to the plastid, even in the presence of brefeldin A. Ligand release at 15 degrees C, which blocks trafficking of Golgi-routed proteins, also allowed significant localization to the plastid. Our data indicate that apicoplast proteins can localize to the region of the plastid when Golgi trafficking is inhibited, but suggest that some steps in import or maturation of the proteins may require a brefeldin A-sensitive GTP exchange factor.

Synthesis of chloroplast galactolipids in apicomplexan parasites.

Monogalactosyldiacylglycerol and digalactosyldiacylglycerol are major chloroplast lipids of algae and land plants and are synthesized within the plastid envelope. Here we report that in Toxoplasma gondii and Plasmodium falciparum lysates, radiolabeled UDP-galactose is incorporated into monogalactosylcerebrosides, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol due to distinct enzymological activities. Furthermore, DGDG is immunologically detected in apicomplexans.