Plasmodium falciparum heat shock protein 70 is able to suppress the thermosensitivity of an Escherichia coli DnaK mutant strain.
Heat shock protein 70 (Hsp 70) and heat shock protein 40 (Hsp 40) are molecular chaperones that ensure that the proteins of the cell are properly folded and functional under both normal and stressful conditions. The malaria parasite Plasmodium falciparum is known to overproduce a heat shock protein 70 (PfHsp 70) in response to thermal stress; however, the in vivo function of this protein still needs to be explored. Using in vivo complementation assays, we found that PfHsp 70 was able to suppress the thermosensitivity of an Escherichia coli dnaK 756 strain, but not that of the corresponding deletion strain (DeltadnaK 52) or dnaK 103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp 70 fused to the substrate-binding domain (SBD) of E. coli DnaK (referred to as PfK), and the ATPase domain of E. coli DnaK coupled to the SBD of PfHsp 70 (KPf). PfK was unable to suppress the thermosensitivity of any of the E. coli strains. In contrast, KPf was able to suppress the thermosensitivity in the E. coli dnaK 756 strain. We also identified two key amino acid residues (V 401 and Q 402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp 70. This is the first example of an Hsp70 from a eukaryotic parasite that can suppress thermosensitivity in a prokaryotic system. In addition, our results also suggest that interdomain communication is critical for the function of the PfHsp 70 and PfHsp 70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp 40 chaperone machinery.
Shonhai
Addmore
A
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa.
Boshoff
Aileen
A
Blatch
Gregory L
GL
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000818:Animals; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D010963:Plasmodium falciparum / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli