The evolution, metabolism and functions of the apicoplast

The malaria parasite, Plasmodium falciparum, harbours a relict plastid known as the ‘apicoplast’. The discovery of the apicoplast ushered in an exciting new prospect for drug development against the parasite. The eubacterial ancestry of the organelle offers a wealth of opportunities for the development of therapeutic interventions. Morphological, biochemical and bioinformatic studies of the apicoplast have further reinforced its ‘plant-like’ characteristics and potential as a drug target. However, we are still not sure why the apicoplast is essential for the parasite's survival. This review explores the origins and metabolic functions of the apicoplast. In an attempt to decipher the role of the organelle within the parasite we also take a closer look at the transporters decorating the plastid to better understand the metabolic exchanges between the apicoplast and the rest of the parasite cell

A MAC Mode for Lightweight Block Ciphers

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Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite <i>Plasmodium falciparum</i>

<div><p>Apicoplasts are vestigial plastids in apicomplexan parasites like <i>Plasmodium</i>, the causative agent of malaria. Apicomplexan parasites are dependant on their apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs) are embedded in the outer apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer apicoplast membrane of the human malaria parasite <i>P</i>. <i>falciparum</i>. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer apicoplast membrane.</p></div

Predicted masses and primer pairs for the <i>Pf</i>oTPT transfection constructs.

<p>The tyrosine (Y) codon in synthetic <i>Pf</i>oTPT and the alanine (A) codon in Y10A are in bold. The predicted mass given for TMD10 is for the entire TMD1+TMD10 fusion protein. The <i>Hind</i>III cloning sites are underlined. A <i>Hind</i>III site is naturally found within TMD1 and one was added to the TMD10 forward primer.</p

Western blots showing that all <i>Pf</i>oTPT modification constructs produce membrane bound proteins of appropriate apparent masses.

<p>The left column shows the insoluble (membrane) fraction while the right column shows the soluble (non-membrane) fractions after Triton X-114 protein partitioning. The apicoplast stromal protein Hsp60 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159603#pone.0159603.ref044" target="_blank">44</a>] is loaded as a soluble protein control, and <i>Pf</i>oTPT is loaded as an insoluble protein control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159603#pone.0159603.ref012" target="_blank">12</a>]. Hsp60 fractions were probed with anti-Hsp60 and all other fractions with anti-HA.</p

Gene expression constructs defining elements of <i>Pf</i>oTPT essential for targeting to the outer membrane of the apicoplast.

<p>TMDs are represented by numbered boxes and are joined by loops. All <i>Pf</i>oTPT constructs are episomally expressed under the <i>Pf</i>CRT promoter and tagged with triple HA at the C-terminus detected with anti-HA and secondary antibody conjugated to FITC (green) in parasites within erythrocytes. Co-localisation of the apicoplast using antisera against apicoplast stromal marker, ACP, is shown in red. Nuclei are stained with Hoechst (blue), and transmitted light images of the parasites within their host red blood cell are shown on the right. Scale bars = 2μm. A. Full length, synthetic <i>Pf</i>oTPT co-localises with ACP. B. Point mutation of tyrosine residue at position 10 (✪) in the synthetic <i>Pf</i>oTPT (Y10A) abrogates targeting to the apicoplast showing no co-localisation with the apicoplast marker. C. Removal of TMD 10 (TMD1to9) abrogates targeting to the apicoplast showing no co-localisation with the apicoplast marker. D. Removal of TMDs 9 and 10 (TMD1to8) abrogates targeting to the apicoplast showing no co-localisation with the apicoplast marker. E. Removal of TMDs 7, 8, 9 and 10 (TMD1to6) abrogates targeting to the apicoplast showing no co-localisation with the apicoplast marker. F. Removal of TMDs 6, 7, 8, 9 and 10 (TMD1to5) abrogates targeting to the apicoplast showing no co-localisation with the apicoplast marker. G. Removal of TMDs 3, 4, 5, 6, 7, 8, 9 and 10 (TMD1to2) abrogates targeting to the apicoplast showing no co-localisation with the apicoplast marker and diffuse staining throughout the parasite. H. Removal of TMDs 2, 3, 4, 5, 6, 7, 8, 9 and 10 (TMD1) abrogates targeting to the apicoplast showing no co-localisation with the apicoplast marker and diffuse staining throughout the parasite. I. A combination of TMD1 and TMD10 (TMD1+TMD10) was not sufficient to reconstitute targeting to the apicoplast, showing no co-localisation with the apicoplast marker.</p

DNA sequence of the <i>Pf</i>oTPT gene from start to stop codon.

<p>The part of the forward primer found in the coding region is shown in black and was used to generate TMD1to9, TMD1to8, TMD1to6, TMD1to5, TMD1to2 and TMD1. The part of the TMD10 forward primer matching the coding region is shown in purple. Reverse primers used to amplify portions of the gene containing various TMDs are shown in red. The tyrosine codon is shown in orange, the <i>Hind</i>III cloning site is shown in green and loop 9, included in TMD1+TMD10, is shown in blue.</p