Abstract
The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ32, which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ32 facilitates RB43 bacteriophage proliferation is discussed.
Highlights
The universally conserved molecular chaperone machines maintain cellular protein homeostasis by acting at almost every stage in the life of proteins [1]
We show that the ORFan gene 057w from the T4-related enterobacteriophage RB43, encodes a bona fide type III DnaJ-like protein, named Rki, which interacts with the E. coli host multifunctional DnaK chaperone
This work shows for the first time that bacteriophages can encode functional J-domain proteins capable of hijacking the host Hsp70 chaperone to facilitate viral proliferation
Summary
The universally conserved molecular chaperone machines maintain cellular protein homeostasis by acting at almost every stage in the life of proteins [1]. In the bacterium Escherichia coli, the multifunctional DnaK (Hsp70) chaperone machine (the DnaK/ DnaJ/GrpE complex) performs key cellular functions under both physiological and stress conditions [2,3,4]. It assists de novo protein folding and targeting to biological membranes, protein quality control, assembly or disassembly of oligomeric complexes, and signal transduction. All the DnaJ/Hsp cochaperones are characterized by the presence of a compact domain of about 70 amino acids, called the J-domain, which is essential for a functional interaction with Hsp70 These cochaperones are generally called JDPs for J-domain proteins [14]. Type II JDPs generally have a similar domain arrangement except that they do not possess a
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