Trafficking of the exported P. falciparum chaperone PfHsp70x.

Manuel Rhiel,Cecilia Sanchez,Christine Schaeffer-Reiss,Sarah C Charnaud,Jude M Przyborski,Simone Külzer,Sebastian Müller,Verena Bittl,Benoit Westermann,Paul R Gilson,Michael Lanzer,Anke Tribensky,Maja Strecker,Brendan S Crabb

doi:10.1038/srep36174

Abstract

Plasmodium falciparum extensively modifies its chosen host cell, the mature human erythrocyte. This remodelling is carried out by parasite-encoded proteins that are exported into the host cell. To gain access to the human red blood cell, these proteins must cross the parasitophorous vacuole, a membrane bound compartment surrounding the parasite that is generated during the invasion process. Many exported proteins carry a so-called PEXEL/HT signal that directs their transport. We recently reported the unexpected finding of a species-restricted parasite-encoded Hsp70, termed PfHsp70x, which is exported into the host erythrocyte cytosol. PfHsp70x lacks a classical PEXEL/HT motif, and its transport appears to be mediated by a 7 amino acid motif directly following the hydrophobic N-terminal secretory signal. In this report, we analyse this short targeting sequence in detail. Surprisingly, both a reversed and scrambled version of the motif retained the capacity to confer protein export. Site directed mutagenesis of glutamate residues within this region leads to a block of protein trafficking within the lumen of the PV. In contrast to PEXEL-containing proteins, the targeting signal is not cleaved, but appears to be acetylated. Furthermore we show that, like other exported proteins, trafficking of PfHsp70x requires the vacuolar translocon, PTEX.

Highlights

Plasmodium falciparum is responsible for the most serious form of human malaria, which causes over 0.6 million deaths annually
We have previously shown that a reporter protein containing the first 32 N-terminal amino acids of PfHsp70x fused to GFP is partially exported to the host cell cytosol, mirroring the distribution of the endogenous full-length protein[19]
Since the discovery that malaria parasites synthesise and traffic their own proteins to and through the infected host-erythrocyte, much research effort has been focused on understanding this unusual novel protein trafficking system, including analysis of the signals, mechanisms and machinery required for high-fidelity delivery of proteins to specific sub-cellular compartments

Summary

Introduction

Plasmodium falciparum is responsible for the most serious form of human malaria, which causes over 0.6 million deaths annually. A major breakthrough in our understanding of protein export in malaria parasites came with the discovery that many exported parasite proteins contain an N-terminal conserved trafficking signal, referred to as PEXEL or HT4,5,7 Mutation of this motif leads to a block in protein transport to the host cell. We have previously identified an exported Hsp[70] (PfHsp70x) in P. falciparum-infected erythrocytes[19] This protein contains an N-terminal secretory signal sequence, lacks a PEXEL/HT signal, but is partially exported to the host erythrocyte and can be regarded as a PNEP. Trafficking of PfHsp70x to the host cell appears to be driven by the 32 N-terminal amino acids, including the signal sequence, upstream of the highly conserved Hsp[70] ATPase domain (Fig. 1B)[19]. Our study represents the first to finely decipher the trafficking information necessary for export of a soluble PNEP to the P. falciparum-infected human erythrocyte and to elucidate the export pathway

Methods

Results

Conclusion