Caught on camera: a snapshot of proteins moving through malaria-infected erythrocytes

Abstract

The intracellular development of malaria parasites and the transport of molecules to the cytoplasm and surface of red blood cells (RBCs) presents the biophysical challenge of crossing three membranes. This complicated export of parasite-encoded proteins to the RBC surface is important for the pathogenicity of the malaria parasite, as the development of knob-like structures is known to facilitate cytoadhesion and sequestration and, ultimately, evasion of parasite killing by the spleen.To better understand this process, Wickham et al.1xTrafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes. Wickham, M.E. et al. EMBO J. 2001; 20: 5636–5649Crossref | PubMed | Scopus (211)See all References1 fused a protein known to be targeted to the RBC surface – knob-associated histidine-rich protein (KAHRP) – to green fluorescent protein (GFP), and followed its pathway of transport using fluorescence microscopy and fluorescent recovery after photobleaching (FRAP). These techniques allowed them to study the movement of molecules in cells in real time.As the first step, they determined the peptide sequences required to target KAHRP to the RBC surface by using two different GFP-fusion constructs, one containing the first 60 amino acids of KAHRP fused to GFP, and another containing the first 123 amino acids of KAHRP fused to GFP. In transfected malaria parasites, the first construct demonstrated a ‘necklace of beads’ around the parasite, suggesting that the fusion protein was being accumulated in the parasitophorous space. This observation suggests that the first 60 amino acids were sufficient to allow (1) entry into the secretory pathway and targeting to the parasite plasma membrane; and (2) secretion from the parasite into the parasitophorous vacuolar space, but insufficient for targeting of the protein across the parasitophorous vacuole membrane and into the RBC cytoplasm. The GFP-fusion containing the first 123 amino acids, which encompasses the histidine-rich domain of KAHRP, was successfully targeted to the RBC cytoplasm, indicating that the following 63 amino acids were sufficient for targeting to the host cell membrane. The trafficking of these GFP-fusion proteins was also sensitive to Brefeldin A, which indicated that the proteins were moving through the endoplasmic reticulum (ER). Moreover, this finding suggests the involvement of a classical vesicle-mediated pathway rather than an alternative pathway, as previously suggested.The most striking observation in this study was the finding that GFP-fused KAHRP associated with structures in the RBC cytoplasm called Maurer's clefts. The function of these structures has been unclear and it has also been a mystery from where they are derived. In addition to the GFP-fusion proteins, two other knob-associated proteins, as well as an ER-resident protein known to be involved in ER-to-Golgi budding (SAR1p), co-localized to these Maurer's clefts. The association of the GFP-fusion proteins with these structures strongly suggests that the Maurer's clefts are derived from the parasite and, by some elaborate mechanism, they are relocated out of the parasite and into the host cell cytoplasm. This type of elaboration of the secretory system of an intracellular pathogen is the first example of its kind in eukaryotic cell biology.