Heat-inactivated Proteins Managed by DnaKJ-GrpE-ClpB Chaperones Are Released as a Chaperonin-recognizable Non-native Form

Abstract

Chaperones of Thermus thermophilus cooperate in reactivation of heat-inactivated proteins. The protein, inactivated at a high temperature in a TDnaKJ-GrpE set, recovered its activity during subsequent incubation with TClpB at moderate temperature (Motohashi, K., Watanabe, Y., Yohda, M., and Yoshida, M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7184 -7189). Here, we report that the addition of chaperonin (Tcpn) at moderate temperature improves the yield of the TDnaKJ-GrpE-ClpBdependent reactivation. The trap-Tcpn, which binds substrate protein but does not release it, inhibits reactivation severely. Maximum recovery is gained at substoichiometric amounts of each component of TDnaKJ, TGrpE, and TClpB relative to the substrate monomer. These observations indicate that, driven by ATP hydrolysis, TDnaKJ-GrpE-ClpB chaperones catalytically cooperate and release heat-inactivated protein as a non-native, chaperonin-recognizable folding intermediate.Recently, the functional chaperone cooperation between Hsp70 and Hsp104 in reactivation of heat-inactivated proteins has been noticed. In eukaryotes, this cooperation enables yeast to recover from heat stress in vivo (1, 2) and reactivate proteins that had been chemically denatured and allowed to aggregate in vitro (3). In prokaryotes, we have demonstrated that heatinactivated proteins were rescued by the cooperation between DnaK (Hsp70)-DnaJ-GrpE and ClpB (Hsp104) from a thermophilic eubacteria, Thermus thermophilus (4). Such a cooperation was also reported for Escherichia coli (5). Here, we report the role of each component in this cooperation and relationship to Hsp60 (GroEL, chaperonin).E. coli DnaK, together with its co-chaperones, DnaJ and GrpE, can assist in the refolding of some proteins (6 -8). The binding and release of substrate proteins are regulated by ATP/ADP state of DnaK, which is under tight control with DnaJ and GrpE (9 -11); DnaJ accelerate the rate of ATP hydrolysis and GrpE promotes the ADP/ATP exchange (12). Unlike in other bacteria, DnaK and DnaJ are purified only as a stable trigonal ring complex from T. thermophilus (13 As a member of the Hsp104 family, bacterial ClpB has two nucleotide binding domains, each displaying one set of consensus ATP-binding sequence motifs, and forms a homohexameric complex (18 -21). Like E. coli ClpB, TClpB (97 kDa) has an ATPase activity that is stimulated by the addition of a model denatured protein (4,22). TClpB shows chaperone activity only when combined with TDnaKJ complex and TGrpE. When thermophilic lactate dehydrogenase (LDH) is heat-inactivated at high temperature in the presence of the KJE set and ATP, the enzyme activity of LDH is recovered during subsequent incubation with TClpB at a moderate temperature (4).Chaperonin (Hsp60) is another chaperone that plays a critical role in cells, even under condition without stress (23). E. coli chaperonin, GroEL, is composed of 14 identical subunits forming two heptamer rings stacked back to back (24). GroEL binds nonnative protein and sequesters it into the central cavity...

Trigonal DnaK-DnaJ Complex Versus Free DnaK and DnaJ

“…In eukaryotes, this cooperation helps yeast cells to recover from severe heat stress (8 -10), and in vitro experiments have shown that HSP104-HSP70 cooperation facilitates reactivation of the proteins that had been chemically denatured and aggregated (3). In prokaryotes, as we have demonstrated for chaperones from a thermophilic eubacteria, Thermus thermophilus, heatdamaged proteins were rescued by the cooperative chaperone functions of ClpB (bacterial HSP104), DnaK (bacterial HSP70), DnaJ, and GrpE (4,11). ClpB forms a homo-hexameric complex (580 kDa) as an active species and needs ATP hydrolysis for the function (12)(13)(14)(15)(16)(17).…”

“…TDafA is a small protein necessary to assemble TDnaK and TDnaJ into K⅐J (20); TGrpE (22 kDa) is isolated as a homodimer (21)(22)(23). The stable K⅐J has been reported only for DnaK and DnaJ from T. thermophilus, but the chaperone function of K⅐J has been studied with a general interest because the current model for the mechanism of DnaK function assumes only transient but not stable interaction between DnaK and DnaJ (4,11,16,19,23). Related to this contention, the chaperone activity of uncomplexed TDnaK and TDnaJ (termed KϩJ) has been reported (14,23,24), but the difference from that of K⅐J is not clear.…”

α-Crystallin-Type Heat Shock Proteins: Socializing Minichaperones in the Context of a Multichaperone Network

“…Only recently, it was recognized that certain chaperones are able to solubilize aggregated proteins in vivo. The concerted action of Hsp104, Hsp70, and Hsp40 in yeast (92) or of ClpB and DnaK in E. coli (94,211,214,347) recycles precipitated proteins into the folding pathway. Disaggregated material is released in a nonnative form that either can refold spontaneously or can be further processed by cellular chaperones.…”

The ClpB Homolog Hsp78 Is Required for the Efficient Degradation of Proteins in the Mitochondrial Matrix

“…Since a similar activity had previously been described for the cytosolic Hsp104 (10,11), the current view is that proteins of the ClpB subtype are mainly required for the protection of cellular protein function under severe stress conditions (7,12,13). Evidently, cellular proteins can be solubilized from heat-induced aggregates by ClpB-type chaperones and are released in a state that is recognized and stabilized by the Hsp70 system, which promotes further folding to the native conformation (8,11,14). A similar role in thermotolerance and stress protection has been suggested for the mitochondrial homolog Hsp78 (15,16).…”