200 Fig. 5 PfHop, PfHsp70, PfHsp90 and PfJ4 may associate in a heterologous complex. a The presence of PfHop, PfHsp70, PfHsp90 and PfJ4 in the elution fractions obtained by size exclusion chromatography was determined by Western analysis using corresponding antibodies. Approximate molec- ular weights (in kDa) associated with select fractions are given (top panel). b Co- immunoprecipitation of PfHop, PfHsp70 and PfHsp90 using anti-PfHop antibodies, in the absence and presence of ATP. PfHop and immunoglobulin heavy chain are shown by the arrows. The results are repre- sentative of at least three inde- pendent experiments G.W. Gitau et al. further mirror the complexity of the mechanism by which the Hsp70–Hop–Hsp90 pathway is governed. For example, a non-canonical Hop protein with a missing TPR1 domain from Caenorhabditis elegans (CeHop) has been described as capable of binding both Hsp70 and Hsp90 through the TPR2A domain (Gaiser et al. 2009). However, this essential protein could not bind both Hsp70 and Hsp90 at once. This could suggest that Hop may independently associate with Hsp90 and Hsp70. Interestingly, none of the fractions we obtained had PfHsp90 and PfHop in the absence of PfHsp70, in spite of the fact that Hsp90 is known to interact with Hop in the absence of Hsp70 (Hernández et al. 2002b). This may be due to the transient and dynamic nature of the association between PfHsp90 and PfHop since the immunofluorescence data suggested a strong associa- tion between PfHsp90 and PfHop (Fig. 4b). Previous studies proposed that Hsp40 co-chaperones (belonging to the type I and type II subfamilies; Cheetham and Caplan 1998) are involved in the assembly of the Hsp70–Hop–Hsp90 complex (Cintron and Toft 2006). In this study, we sought to investigate if PfJ4, a type II Hsp40 known to associate with PfHsp70 (Pesce et al. 2008), would occur in the PfHsp70–PfHop–PfHsp90 complex. PfJ4 was not eluted in the fraction in which both PfHsp70 and PfHsp90 occurred (Fig. 5a). This suggests that PfJ4 may not be involved in the formation of this complex. We recently described a type I Hsp40 from P. falciparum (PfHsp40) that functionally co-operates with PfHsp70 in vitro (Botha et al. 2011). PfHsp40 may possibly be one of the P. falciparum Hsp40 proteins that are involved in the formation of the PfHsp70–PfHop complex, but this possibility still needs to be experimentally verified. Based on co-immunoprecipitation analysis, PfHsp70 associated with both PfHsp90 and PfHop (Fig. 5b). As expected, in the absence of added ATP, PfHsp70 interacted with more PfHop protein than in the presence of added ATP (Fig. 5b). This is in agreement with a previous study that proposed that Hop binds to Hsp70 when the latter is