Trans Expression of a Plasmodium falciparum Histidine-rich Protein II (HRPII) Reveals Sorting of Soluble Proteins in the Periphery of the Host Erythrocyte and Disrupts Transport to the Malarial Food Vacuole

Thomas Akompong,Madhusudan Kadekoppala,Travis Harrison,Anna Oksman,Daniel E Goldberg,Hisashi Fujioka,Benjamin U Samuel,David Sullivan,Kasturi Haldar

doi:10.1074/jbc.m201968200

Abstract

The heme polymer hemozoin is produced in the food vacuole (fv) of the parasite after hemoglobin proteolysis and is the target of the drug chloroquine. A candidate heme polymerase, the histidine-rich protein II (HRPII), is proposed to be delivered to the fv by ingestion of the infected-red cell cytoplasm. Here we show that 97% of endogenous Plasmodium falciparum (Pf) HRPII (PfHRPII) is secreted as soluble protein in the periphery of the red cell and avoids endocytosis by the parasite, and 3% remains membrane-bound within the parasite. Transfected cells release 90% of a soluble transgene PfHRPIImyc into the red cell periphery and contain 10% membrane bound within the parasite. Yet these cells show a minor reduction in hemozoin production and IC(50) for chloroquine. They also show decreased transport of resident fv enzyme PfPlasmepsin I, the endoplasmic reticulum (ER) marker PfBiP, and parasite-associated HRPII to fvs. Instead, all three proteins accumulate in the ER, although there is no defect in protein export from the parasite. The data suggest that novel mechanisms of sorting (i) soluble antigens like HRPII in the red cell cytoplasm and (ii) fv-bound membrane complexes in the ER regulate parasite digestive processes.

Highlights

The heme polymer hemozoin is produced in the food vacuole of the parasite after hemoglobin proteolysis and is the target of the drug chloroquine
Effects of PfHRPIImyc Expression on Hemozoin Production and Chloroquine IC50—Because PfHRPII is proposed to be a heme polymerase, we investigated whether hemozoin levels as well as chloroquine sensitivity were altered in transfected cells
Our studies suggest that both PfHRPII and PfHRPIImyc are stable proteins that are exported to the red cell and the fv. 3– 4-Fold elevation of PfHRPIImyc expression results in a corresponding increase in total histidine-rich protein II (HRPII) release into the red cells but no increase of HRPII protein in the fv

Summary

Introduction

The heme polymer hemozoin is produced in the food vacuole (fv) of the parasite after hemoglobin proteolysis and is the target of the drug chloroquine. Histidine-rich proteins have been shown to function as heme polymerases in vitro, and the best characterized of these proteins is P. falciparum HRPII (or PfHRPII) [11,12,13] This protein has been detected in the red cell as well as the fv, leading to the suggestion that it is ingested from the red cell by the cytostome along with hemoglobin and subsequently delivered to the fv. Studies suggest that resident fv proteases synthesized in a membrane-bound pro-form are transported to the PVM and retrieved into the newly developing cytostome, which ingests hemoglobin [11, 14] This has rendered attractive a general model that both membrane and lumenal components of food vacuole may be delivered to the PV and the red cell from where they are internalized into the fv [5].

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