Construction of a Plasmodium falciparum Rab-interactome identifies CK1 and PKA as Rab-effector kinases in malaria parasites

Background information The pathology causing stages of the human malaria parasite Plasmodium falciparum reside within red blood cells that are devoid of any regulated transport system. The parasite, therefore, is entirely responsible for mediating vesicular transport within itself and in the infected erythrocyte cytoplasm, and it does so in part via its family of 11 Rab GTPases. Putative functions have been ascribed to Plasmodium Rabs due to their homology with Rabs of yeast, particularly with Saccharomyces that has an equivalent number of rab/ypt genes and where analyses of Ypt function is well characterized. Results Rabs are important regulators of vesicular traffic due to their capacity to recruit specific effectors. In order to identify P. falciparum Rab (PfRab) effectors, we first built a Ypt-interactome by exploiting genetic and physical binding data available at the Saccharomyces genome database (SGD). We then constructed a PfRab-interactome using putative parasite Rab-effectors identified by homology to Ypt-effectors. We demonstrate its potential by wet-bench testing three predictions; that casein kinase-1 (PfCK1) is a specific Rab5B interacting protein and that the catalytic subunit of cAMP-dependent protein kinase A (PfPKA-C) is a PfRab5A and PfRab7 effector. Conclusions The establishment of a shared set of physical Ypt/PfRab-effector proteins sheds light on a core set Plasmodium Rab-interactants shared with yeast. The PfRab-interactome should benefit vesicular trafficking studies in malaria parasites. The recruitment of PfCK1 to PfRab5B+ and PfPKA-C to PfRab5A+ and PfRab7+ vesicles, respectively, suggests that PfRab-recruited kinases potentially play a role in early and late endosome function in malaria parasites

The Plasmodium falciparum Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance

Resolving the homology-function relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology

With advances in DNA sequencing technology, it is increasingly common and tractable to informatically look for genes of interest in the genomic databases of parasitic organisms and infer cellular states. Assignment of a putative gene function based on homology to functionally characterized genes in other organisms, though powerful, relies on the implicit assumption of functional homology, i.e. that orthology indicates conserved function. Eukaryotes reveal a dazzling array of cellular features and structural organization, suggesting a concomitant diversity in their underlying molecular machinery. Significantly, examples of novel functions for pre-existing or new paralogues are not uncommon. Do these examples undermine the basic assumption of functional homology, especially in parasitic protists, which are often highly derived? Here we examine the extent to which functional homology exists between organisms spanning the eukaryotic lineage. By comparing membrane trafficking proteins between parasitic protists and traditional model organisms, where direct functional evidence is available, we find that function is indeed largely conserved between orthologues, albeit with significant adaptation arising from the unique biological features within each lineage

Caractérisation des GTPases Rab5 de Plasmodium

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

PfRab5B colocalises with PfMSP1 and PfCK1, but not with haemoglobin.

<p>(A) PfRab5A colocalises with haemoglobin (HBA1) containing vesicles (r = 0.724), unlike PfRab5B (r = 0.081; n = 3). (B) PfRab5B colocalises to differing degrees (r = 0.849; n = 3) with the C-terminal 19 kDa fragment of PfMSP1 on structures close to the food vacuole and the parasite nucleus shown in blue by DAPI staining. (C) PfRab5B colocalises with PfCK1 on intracellular structures (r = 0.615; n = 3) and at the parasite plasma membrane (r = 0.475; n = 3). Areas of colocalistaion are shown in white and used to calculate Pearson's r coefficients. Scale bars, 2 µm.</p

Recombinant PfRab5B is N-myristoylated by PfNMT <i>in vitro</i>.

<p>PfRab5B and the G2A and G2AC3A variants were incubated with [³H]-myristoyl CoA in the presence of N-myristoyl transferase. Recombinant ARF and ARFG2A were treated in the same way, acting as controls. N-myristoylation was detected by the incorporation of radiolabel into the substrate following SDS-PAGE and fluorography and the upper band at 50 kDa is due to label binding to PfNMT. Both PfRab5B and ARF migrated with mobility slightly faster than the 25 kDa molecular mass marker. No incorporation was detected if the N-terminal glycine residue of either protein was replaced with alanine (G2A).</p

Rab5 Isoforms Specifically Regulate Different Modes of Endocytosis in Leishmania

Pathway between enclosure all on the right and the tower area, with pergola arching overhead; Roluos is a modern Cambodian small town and an archeological site about 13 km east of Siem Reap. Once the seat of Hariharalaya, the first capital of the Khmer Empire north of Tonlé Sap. Among the "Rolous Group" of temples is the most impressive, the Bakong complex, whose central pyramid stands 14 meters tall. Constructed by the third Angkorian-era king Indravarman I as his state-temple (to house the royal liṅga Indreshvara), Bakong represents the first reasonably complete temple-mountain architectural formula on a grand scale and set the architectural tone for the next 400 years. The temple displays a very early use of stone rather than brick (brick was used in the 22 smaller satellite tower-sanctuaries between the two moats.) Bakong received additions and was expanded by later kings. The uppermost section and tower were added in the 12th century. Source: Grove Art Online; http://www.oxfordartonline.com/ (accessed 4/29/2013