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

Abstract

Background informationThe 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.ResultsRabs 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.ConclusionsThe 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.

Isoprenoid Biosynthesis Inhibition Disrupts Rab5 Localization and Food Vacuolar Integrity in Plasmodium falciparum

“…Although casein kinase physically associates with Rab5b, the downstream effectors of Rab5s in malaria are not known (30). A single essential, wortmannin-sensitive PI3-K has been described for P. falciparum, which can synthesize PI(3)P, PI(3,4)P 2 , and PI(3,4,5)P 3 (35,37).…”

“…Fixed parasites were permeabilized, blocked, and incubated with antibodies, as previously described (29). Rabbit polyclonal antibodies against recombinant P. falciparum Rab5a (Pf-Rab5a) and Pf-Rab5c were kindly provided by Gordon Langsley (Institut Cochin, France) (30) and were used at 1:500 and 1:1,000 dilutions, respectively. The secondary antibody used was Alexa Fluor 488 goat anti-rabbit IgG(HϩL) (catalog number A11008; Invitrogen) at a 1:500 dilution, and Hoechst 33258 was used as a nuclear counterstain.…”

Exploring the Plasmodium falciparum cyclic-adenosine monophosphate (cAMP)-dependent protein kinase (PfPKA) as a therapeutic target

“…Analyses of PfPKA-R for ordered regions predict that the N-terminus is highly disordered making it accessible to many modifications. Some of these sites may introduce a docking site for another protein such as parasite 14-3-3 that was recently shown to be a substrate for PKA [52], or for Rab7 that has been shown to bind PfPKA-C [53]. Clearly, N-terminal binding of non-AKAP proteins could introduce an alternative mechanism for R subunit dimerization.…”

Atypical Mitogen-Activated Protein Kinase Phosphatase Implicated in Regulating Transition from Pre-S-Phase Asexual Intraerythrocytic Development of Plasmodium falciparum

“…There is growing evidence that kinases are critical regulators of cell growth and development in Plasmodium species (7)(8)(9). Plasmodium kinases, for example, have been found to be involved with the initial invasion of host cells in addition to egress and differentiation (6,10).…”