Abstract
Using the native proteins lambda P, lambda O, delta 32, and RepA, as well as permanently unfolded alpha-carboxymethylated lactalbumin, we show that DnaK and DnaJ molecular chaperones possess differential affinity toward these protein substrates. In this paper we present evidence that the DnaK protein binds not only to short hydrophobic peptides, which are in an extended conformation, but also efficiently recognizes large native proteins (RepA, lambda P). The best substrate for either the DnaK or DnaJ chaperone is the native P1 coded replication RepA protein. The native delta 32 transcription factor binds more efficiently to DnaJ than to DnaK, whereas unfolded alpha-carboxymethylated lactalbumin or native lambda P binds more efficiently to DnaK than to the DnaJ molecular chaperone. The presence of nucleotides does not change the DnaJ affinity to any of the tested protein substrates. In the case of DnaK, the presence of ATP inhibits, while a nonhydrolyzable ATP analogues markedly stimulates the binding of DnaK to all of these various protein substrates. ADP has no effect on these reactions. In contrast to substrate protein binding, DnaK binds to the DnaJ chaperone protein in a radically different manner, namely ATP stimulates whereas a nonhydrolyzable ATP analogue inhibits the DnaK-DnaJ complex formation. Moreover, the DnaKc94 mutant protein lacking 94 amino acids from its C-terminal domain, which still possesses at ATPase activity and forms a transient complex with protein substrates, does not interact with DnaJ protein. We conclude that the DnaK-ADP form, derived from ATP hydrolysis, possesses low affinity to the protein substrates but can efficiently interact with DnaJ molecular chaperone.
Highlights
The highly conserved and ubiquitous 70-kDa heat shock proteins (Hsp70), or the prokaryotic DnaK homologues, function as molecular chaperones in a variety of cellular processes (reviewed by Gething and Sambrook (1992), Hendrick and Hartl (1993), Georgopoulos et at. (1994), and Craig et at. (1994))
Most models addressing the question of how Hsp70 functions in protein transport or folding assume that Hsp70 bound to ADP (Hsp70-ADP) is the form exhibiting the highest affinity toward the substrate and ATP hydrolysis is required only for the regeneration of such a form (for review, see Craig et at. (1994))
It is generally assumed that the Hsp70 class of protein binds to unfolded proteins, recognizing short polypeptides that are in an extended configuration
Summary
The highly conserved and ubiquitous 70-kDa heat shock proteins (Hsp70), or the prokaryotic DnaK homologues, function as molecular chaperones in a variety of cellular processes (reviewed by Gething and Sambrook (1992), Hendrick and Hartl (1993), Georgopoulos et at. (1994), and Craig et at. (1994)). The binding and hydrolysis of ATP results in conformational changes to the various Hsp family members (Liberek et al, 1991b; Palleros et al, 1992; Banecki et al, 1992). Most models addressing the question of how Hsp functions in protein transport or folding assume that Hsp bound to ADP (Hsp70-ADP) is the form exhibiting the highest affinity toward the substrate and ATP hydrolysis is required only for the regeneration of such a form This conclusion was based on the findings that Hsp family members form a stable complex with various substrates
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