192 Introduction Heat shock proteins occur in all known life forms, and their main role is to facilitate the folding of other proteins both under normal and stressful conditions. Consequently, some heat shock proteins are induced by cellular stress, thus protecting cells against adverse effects. The role of heat shock proteins in the survival of intracellular parasites extends beyond maintenance of proteostasis, as they are also implicated in the development and pathogenesis of these organisms (reviewed by Shonhai et al. (2011). The aetiological agent of malaria tropica, Plasmodium falciparum, develops and thrives under physiologically divergent conditions throughout its life stages. Heat shock proteins are thought to play an important role in the development of P. falciparum as well as other human parasites (Sharma 1992; Shonhai et al. 2007; Shonhai et al. 2011). Heat shock protein 70 (Hsp70) and Hsp90 are some of the most studied molecular chaperones, proteins which themselves are responsible for the folding of other proteins in the cell. Hsp70 binds non-native proteins whilst substrates of Hsp90 are usually in native-like forms (Wegele et al. 2006). Proteins that require both Hsp70 and Hsp90 to fold are thus transferred from Hsp70 to Hsp90 during the folding process. Eukaryotic Hsp90 participates in the conformational regulation of signal transduction molecules, such as tyrosine kinases and steroid hormone receptors. For example, steroid hormone receptors associate with Hsp90 in order for them to adopt conformational competence for hormone binding (Dittmar and Pratt 1997). In eukaryotes, the essential interaction between Hsp70 and Hsp90 is mediated by the Hsp70–Hsp90 organising protein (Hop; Nicolet and Craig 1989). Both Hsp70 and Hsp90 possess C-terminally located EEVD motifs that interact with Hop via its tetratricopeptide repeat (TPR) domains, TPR1 and TPR2A motifs, respectively (Scheufler et al. 2000). It is most likely that the Hsp70–Hsp90 functional partnership in Plasmodium spp. facilitates the folding of key proteins in the parasite cell, possibly those involved in signal transduction. PfHsp90 is known to play an essential role in the survival of the parasite and the antibiotic geldanamycin is known to inhibit its function (Banumathy et al. 2003). Of the six Hsp70-like proteins encoded by the P. falciparum genome, only the cytosol-nuclear localised chaperone, PfHsp70 possess the EEVD motif (Shonhai et al. 2007) that is crucial for interaction between Hsp70 and Hop. PfHsp90 occurs in the cytosol and migrates to the nucleus in response to heat stress (Acharya et al. 2007). Thus, PfHsp70 and possibly PfHsp90 possibly cooperate in the parasite cell. Although a Hop homologue (PF14_0324) has been identified in the P. falciparum genome (Acharya et al. 2007), its G.W. Gitau et al. function has not been characterised. Banumathy et al. (2003) observed the occurrence of PfHsp90 and PfHsp70 in functional units that were complexed to two other species of proteins with a molecular mass of around 50 and 60 kDa, respectively. Although not directly verified by experimental data, the authors proposed that the two proteins associating with PfHsp70 and PfHsp90 were P. falciparum cyophilin and PfHop. In addition, at least one TPR-rich protein, PP5 phosphatase from P. Falciparum, has been shown to interact with PfHsp90 (Dobson et al. 2001; Kumar et al. 2003). So far. there is no direct experimental evidence for the existence of a functional PfHsp70–PfHsp90 partnership. However, the presence of PfHsp90 and PfHsp70 in common cellular compartments, coupled to data based on predictive bioinformatics suggest that the two chaperones may functionally interact through a PfHop-mediated path- way (Pavithra et al. 2004). The possible existence of a Hop- mediated Hsp70–Hsp90 partnership in P. falciparum is important, given the essential roles of these proteins. Indeed, this pathway has been proposed as a potential anti-malarial drug target (Pesce et al. 2010; Shonhai 2010). The aim of the current study was to investigate the role of PfHop as a potential mediator of the PfHsp70–PfHsp90 pathway. We cloned, expressed and purified recombinant PfHop protein to facilitate its further characterisation. We further analysed the cellular localisation of PfHop and its association with PfHsp70 and PfHsp90. Materials and methods Bioinformatics To map out the TPR domains in PfHop, Clustal W alignments of Hop homologues from Plasmodium species, human, mouse and yeast were performed utilizing the Bioedit program version 7.0.5.3 (Hall 1999). A three-dimensional model of PfHop was generated by SWISS-MODEL (Arnold et al. 2006; Guex and Peitsch 1997; Schwede et al. 2003). The images were subsequently visualised using the PyMol molecular graphics system, version 0.99rc6 (DeLano 2002). Peptide-directed PfHop and PfHsp90 antibody design and synthesis Anti-peptide antibodies specific to PfHop (PlasmoDB accession number: PF14_0324) and PfHsp90 (PlasmoDB accession number, PF07_0029) were generated commercially (Eurogentec) by immunisation of rabbits with the following synthetic peptides: TGEGNDAEERQRQQR, corresponding to amino acids 195–206 of the PfHop sequence and a mixture of two peptides (CIRYESITDTQKLSAE and CPKRAPFDM