Molecular & Biochemical Parasitology 174 (2010) 66–69 Contents lists available at ScienceDirect Molecular & Biochemical Parasitology Short communication Localization of apical sushi protein in Plasmodium falciparum merozoites Anand Srivastava 1 , Shailja Singh, Shikha Dhawan, M. Mahmood Alam, Asif Mohmmed, Chetan E. Chitnis ∗ Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110067, India article info Article history: Received 4 March 2010 Received in revised form 1 June 2010 Accepted 2 June 2010 Available online 9 June 2010 Keywords: Plasmodium falciparum Apical sushi protein Rhoptry neck protein abstract Plasmodium falciparum belongs to the Apicomplexan group of parasites and is characterised by presence of specialized secretory organelles at the apical end. These apical organelles, referred to as microneme and rhoptries, contain proteins that play important roles during host cell invasion by mediating specific func- tions such as initial attachment, apical reorientation and junction formation. Recently, a protein referred to as P. falciparum apical sushi protein (PfASP), which is expressed at late schizont stage, was localized to micronemes of P. falciparum merozoites. In the present study, we have used indirect immunofluorescence assays and immunoelectron microscopy to demonstrate that PfASP is localized in the neck of rhoptries and not in micronemes as previously described. © 2010 Elsevier B.V. All rights reserved. Malaria remains one of the important and most widespread par- asitic diseases. It is responsible for over one million deaths per year, mostly in sub-Saharan Africa [1]. Malaria parasites belong to Api- complexan group of parasites and harbour specialized secretory organelles referred to as rhoptries and micronemes at the apical end. During development of blood stage schizonts various pro- teins needed for initial attachment, reorientation and invasion into erythrocyte get compartmentalized into these apical organelles [2]. Previous studies have shown that micronemes contain sev- eral proteins including apical membrane antigen-1 (AMA-1) [3] and erythrocyte binding antigens such as EBA-175, EBA-140 and EBA-180 [4,5]. The rhoptries consist of two distinct parts: an elec- tron dense rounded basal bulb and a less dense rhoptry neck that ends just beneath the plasma membrane at the apical promi- nence [6]. Plasmodium falciparum rhoptries contain different sets of proteins in the basal bulb and neck. The P. falciparum high molecular mass rhoptry protein (PfRhopH) complex comprising of three distinct gene products namely, RhopH1(CLAG3.1), RhopH2 and RhopH3 is present in the rhoptry bulb [7,8]. Rhoptry neck protein (PfRON4), a homologue of Toxoplasma gondii rhoptry neck protein TgRON4, is shown to be present at the apical end of the rhoptries [9]. Recently, a GPI anchored merozoite protein, Pf34, and apical asparagine rich protein (PfAARP), were also localized to the rhoptry neck [10,11]. After the release of merozoites from schizonts contents of both rhoptries and micronemes are secreted ∗ Corresponding author. Tel.: +91 11 2674 2895; fax: +91 11 2674 2895. E-mail address: cchitnis@icgeb.res.in (C.E. Chitnis). 1 Current address: Institut Pasteur, Unité de Biologie des Interactions Hôte- Parasite, CNRS URA2581, 25, rue du Docteur Roux, F-75724 Paris Cedex 15, France. to the merozoite surface prior to entry of host cell. We have recently shown that exposure of P. falciparum merozoite to low potassium ion concentrations as found in blood plasma leads to secretion of microneme proteins such as EBA-175 and AMA-1 [12]. Subsequently, interaction of EBA-175 with glycophorin A on the RBC surface provides a signal for release of rhoptry proteins [12]. These data suggest that contents of apical organelles are released in a well defined sequence. Correct localization of proteins in apical organelles is thus important for effective host cell inva- sion. Recently, a protein named as P. falciparum apical sushi protein (PfASP) was identified [13]. Orthologs of PfASP are also found in murine (Plasmodium yoelii and Plasmodium berghei), simian (Plas- modium knowlesi) and human (Plasmodium vivax) malaria parasites [13]. The gene encoding PfASP is 2639 bp long, consists of four exons and is highly up-regulated in schizont stage parasites. PfASP contains an N-terminal signal sequence and a C-terminal trans- membrane domain. The extracellular region of PfASP contains two distinct cysteine rich regions which are conserved in orthologs of PfASP. Based on conserved cysteine-rich regions, PfASP protein was divided into four different regions labelled as regions I–IV (Fig. 1A). Region IV contains a domain that shares homology with “sushi domains” which are characterised by a consensus sequence that spans approximately 60 residues and contains four invariant cysteine residues [14,15]. The sushi domains present in various complement regulator proteins found in mammals and viruses are known to interact with complement components C3b and C4b, interferon- and interleukin-15 [14]. Analysis of the transcription profile of the gene encoding PfASP shows that the PfASP gene is transcribed at late schizont stage [16]. Detection using specific anti- sera suggested that PfASP was localized to micronemes at the apical 0166-6851/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2010.06.003