Cell Stress and Chaperones

, Volume 16, Issue 4, pp 389–401

Plasmodium falciparum encodes a single cytosolic type I Hsp40 that functionally interacts with Hsp70 and is upregulated by heat shock


  • Melissa Botha
    • Biomedical Biotechnology Research Unit, Department of Biochemistry, Microbiology and BiotechnologyRhodes University
  • Annette N. Chiang
    • Department of Biological SciencesUniversity of Pittsburgh
  • Patrick G. Needham
    • Department of Biological SciencesUniversity of Pittsburgh
  • Linda L. Stephens
    • Biomedical Biotechnology Research Unit, Department of Biochemistry, Microbiology and BiotechnologyRhodes University
  • Heinrich C. Hoppe
    • Council for Scientific and Industrial Research
  • Simone Külzer
    • Department of Parasitology, Faculty of BiologyPhilipps University Marburg
  • Jude M. Przyborski
    • Department of Parasitology, Faculty of BiologyPhilipps University Marburg
  • Klaus Lingelbach
    • Department of Parasitology, Faculty of BiologyPhilipps University Marburg
  • Peter Wipf
    • Department of ChemistryUniversity of Pittsburgh
  • Jeffrey L. Brodsky
    • Department of Biological SciencesUniversity of Pittsburgh
  • Addmore Shonhai
    • Department of Biochemistry and MicrobiologyUniversity of Zululand
    • Biomedical Biotechnology Research Unit, Department of Biochemistry, Microbiology and BiotechnologyRhodes University
Original Paper

DOI: 10.1007/s12192-010-0250-6

Cite this article as:
Botha, M., Chiang, A.N., Needham, P.G. et al. Cell Stress and Chaperones (2011) 16: 389. doi:10.1007/s12192-010-0250-6


Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) function as molecular chaperones during the folding and trafficking of proteins within most cell types. However, the Hsp70–Hsp40 chaperone partnerships within the malaria parasite, Plasmodium falciparum, have not been elucidated. Only one of the 43 P. falciparum Hsp40s is predicted to be a cytosolic, canonical Hsp40 (termed PfHsp40) capable of interacting with the major cytosolic P. falciparum-encoded Hsp70, PfHsp70. Consistent with this hypothesis, we found that PfHsp40 is upregulated under heat shock conditions in a similar pattern to PfHsp70. In addition, PfHsp70 and PfHsp40 reside mainly in the parasite cytosol, as assessed using indirect immunofluorescence microscopy. Recombinant PfHsp40 stimulated the ATP hydrolytic rates of both PfHsp70 and human Hsp70 similar to other canonical Hsp40s of yeast (Ydj1) and human (Hdj2) origin. In contrast, the Hsp40-stimulated plasmodial and human Hsp70 ATPase activities were differentially inhibited in the presence of pyrimidinone-based small molecule modulators. To further probe the chaperone properties of PfHsp40, protein aggregation suppression assays were conducted. PfHsp40 alone suppressed protein aggregation, and cooperated with PfHsp70 to suppress aggregation. Together, these data represent the first cellular and biochemical evidence for a PfHsp70–PfHsp40 partnership in the malaria parasite, and furthermore that the plasmodial and human Hsp70–Hsp40 chaperones possess unique attributes that are differentially modulated by small molecules.


AggregationATPaseCodon harmonisationHeat shock proteinMalariaMolecular chaperone



Bovine serum albumin




Dimethyl sulphoxide


Heat shock protein 40


Heat shock protein 70


Horseradish peroxidase




Luria–Bertani media


Malate dehydrogenase


Nickel-nitrilotriacetic acid beads


Phosphate-buffered saline


Phenyl methyl sulphonyl fluoride


Sodium dodecyl sulphate–polyacrylamide gel electrophoresis


Tetramethyl rhodamine isothiocyanate

Supplementary material

12192_2010_250_MOESM1_ESM.doc (46 kb)
Fig. S1Protein sequence alignment of PfHsp40, Pfj1 and type I Hsp40 proteins of eukaryotic and prokaryotic origin. Alignments were performed using type I Hsp40 protein sequences from Homo sapiens (Hdj2, NP_001530.1), Saccharomyces cerevisiae (Ydj1, CAA95937.1), E. coli (DnaJ, P08622.3) and P. falciparum (PfHsp40, PF14_0359/NP_702248.1; and Pfj1, PFD0462w/NP_702750.1). The N-terminal extensions of PfHsp40 and Pfj1, and the C-terminal extension of Pfj1 are shaded in red. The highly conserved J domain is shaded in blue with the conserved HPD, KFK and QKRAA motifs indicated in black boxes. The GF region is shaded in green with the conserved DIF motif highlighted in a black box (Cajo et al. [2006] J. Biol. Chem. 281: 12436–12444). The zinc-finger motifs of the zinc-binding domain (yellow shading) are similarly indicated in black boxes. Residues in bold and indicated by a downward arrow are proposed to be involved in substrate binding (Li and Sha [2005] Biochem. J. 386: 453–460). The CAAX-box motifs of PfHsp40, Ydj1 and Hdj2 are shaded in grey. The region of PfHsp40 used for the generation of peptide-based antibodies is shaded in purple. Conserved identical and similar residues are indicated in the consensus line by asterisks and dots, respectively. The alignment was performed using ClustalW (version 1.83; Chenna et al. [2003] Nucleic Acids Res. 31: 3497–3500) (DOC 46 kb)
12192_2010_250_MOESM2_ESM.doc (883 kb)
Fig. S2Heterologous expression and purification of PfHsp40. Recombinant His6-PfHsp40 was expressed in E. coli XL1-Blue (pQPfHsp40), and purified by nickel affinity chromatography under denaturing conditions. The different stages of the purification process were examined by 10% SDS–PAGE (upper panels). Lane 1, E. coli XL1-Blue (pQPfHsp40) whole cell lysate fraction, 16 h post-induction; 2, E. coli XL1-Blue (pQPfHsp40) soluble fraction after sonication and treatment with urea and PEI; 3, unbound protein fraction (flow-through); 4, wash fraction using 150 mM imidazole; 5, elution fraction using 1 M imidazole, showing recovery of purified recombinant His6-PfHsp40 at 49 kDa. The presence of the His6-PfHsp40 protein in the various samples was verified by western blot analysis using anti-His tag antibodies (lower panels). This experiment was repeated at least three times (DOC 883 kb)

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© Cell Stress Society International 2010