Plasmodium falciparum Rab5B is an N-terminally myristoylated Rab GTPase that is targeted to the parasite's plasma and food vacuole membranes.

Carinne Ndjembo Ezougou,Isabelle Coppens,Anthony A Holder,Sally Black,Hélène Yera,Judith L Green,Shahid M Khan,David K Moss,Fathia Ben-Rached,Jing-Wen Lin,Ellen Knuepfer,Amitabha Mukhopadhyay,Chris J Janse,Gordon Langsley

doi:10.1371/journal.pone.0087695

Abstract

Plasmodium falciparum (Pf) has a family of 11 Rab GTPases to regulate its vesicular transport. However, PfRab5B is unique in lacking a C-terminal geranyl-geranylation motif, while having N-terminal palmitoylation and myristoylation motifs. We show that the N-terminal glycine is required for PfRab5B myristoylation in vitro and when an N-terminal PfRab5B fragment possessing both acylation motifs is fused to GFP and expressed in transgenic P. falciparum parasites, the chimeric PfRab5B protein localizes to the plasma membrane. Upon substitution of the modified glycine by alanine the staining becomes diffuse and GFP is found in soluble subcellular fractions. Immuno-electron microscopy shows endogenous PfRab5B decorating the parasite's plasma and food vacuole membranes. Using reverse genetics rab5b couldn't be deleted from the haploid genome of asexual blood stage P. berghei parasites. The failure of PbRab5A or PbRab5C to complement for loss of PbRab5B function indicates non-overlapping roles for the three Plasmodium Rab5s, with PfRab5B involved in trafficking MSP1 to the food vacuole membrane and CK1 to the plasma membrane. We discuss similarities between Plasmodium Rab5B and Arabidopsis thaliana ARA6, a similarly unusual Rab5-like GTPase of plants.

Highlights

Plasmodium falciparum is a parasite that infects red blood cells (RBC) and causes severe human malaria, a disease that kills approximately 750,000 people per year, mostly children living in tropical Africa [1]
The results showed that PfRab5B is a substrate for P. falciparum N-myristoyl-transferase (PfNMT) and the presence of a glycine at position two is required for PfRab5B myristoylation in vitro, which occurs via peptide bond formation with the a-amino group of the N-terminal glycine
P. falciparum has a small family of 11 P. falciparum Rab (PfRab) and all but PfRab5B possess classical geranyl-geranylation motifs at the Cterminus

Summary

Introduction

Plasmodium falciparum is a parasite that infects red blood cells (RBC) and causes severe human malaria, a disease that kills approximately 750,000 people per year, mostly children living in tropical Africa [1]. For survival inside the RBC the parasite has to both import nutrients and to export metabolic waste products and like other eukaryotes it uses Rab GTPases to regulate vesicular trafficking [2] [3] [4]. Rabs are molecular switches belonging to the Ras-superfamily that are conserved from yeast to humans and regulate in space and time the budding and fusion of intracellular vesicles from donor to acceptor membranes [5]. Rabs from different species display a significant degree of sequence conservation in the GTPbinding domain, but they possess a variable N-terminal sequence responsible for recruitment of effectors and a hyper-variable Cterminal domain. The C-terminal domain confers the specific intracellular location for each Rab and has a motif containing one or two cysteines (CAAX, CC, CXC, CCX, CCXX) necessary for the isoprenylation by geranylgeranyl-transferases that is required for their association with vesicle membranes [11]

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