The effect of nucleotides and mitochondrial chaperonin 10 on the structure and chaperone activity of mitochondrial chaperonin 60

Abstract

Mitochondrial chaperonins are necessary for the folding of newly imported and stress-denatured mitochondrial proteins. The goal of this study was to investigate the structure and function of the mammalian mitochondrial chaperonin system. We present evidence that the 60 kDa chaperonin (mt-cpn60) exists in solution in dynamic equilibrium between monomers, heptameric single rings and doubleringed tetradecamers. In the presence of ATP and the 10 kDa cochaperonin (mt-cpn10), the formation of a double ring is favored. ADP at very high concentrations does not inhibit malate dehydrogenase refolding or ATP hydrolysis by mt-cpn60 in the presence of mt-cpn10. We propose that the cis (mt-cpn60) 14´n uleotide´(mt-cpn10) 7 complex is not a stable species and does not bind ADP effectively at its trans binding site.Keywords: chaperonin; mitochondrial folding; cpn60; cpn10; hsp60.The 60 kDa chaperonins constitute a highly conserved family of proteins found in chloroplasts, mitochondria and eubacteria, which plays an important role in the folding of nascent, translocating and stress-denatured proteins [1]. In yeast mitochondria, chaperonins mediate the folding of both newly imported and stress-denatured proteins, and are essential for the viability of yeast under all conditions [2±5]. Consistent with such an important role, numerous studies have found altered levels of mammalian mitochondrial chaperonin 60 (mt-cpn60) associated with various pathological states [6±11]. Despite the importance of this molecule, few studies have been carried out to understand the details of mammalian mt-cpn60 structure and function. Due to the high level of homology at both the sequence level and functional level, it was commonly assumed that mt-cpn60 was mechanistically similar to its well-studied bacterial counterpart, GroEL. Indeed, the mitochondrial chaperonin system can fold denatured proteins in vitro, with a similar efficiency to GroEL [12,13], and is able to replace the bacterial GroEL in Escherichia coli in vivo [14]. However, a number of significant differences have been reported in the structure of mt-cpn60, which suggest that this eucaryotic chaperonin may have developed a different mechanism of action. While all other chaperonin homologs exist as tetradecamers composed of two seven-membered rings, the mammalian mt-cpn60 has been consistently isolated as a single ring [12,13]. Moreover, mt-cpn60 readily dissociates to monomers in the presence of ATP, low temperatures, and the nonphysiological concentrations that are routinely used for in vitro studies [15]. The functional significance of such instability is unclear. One other interesting difference lies in the fact that mitochondrial cpn60s are functional only with their own 10 kDa cofactor, mitochondrial chaperonin 10 (mt-cpn10) [12,15], whereas the bacterial and chloroplast cpn60 can fold proteins with cpn10 from any source [12,13,16±18].Numerous mechanistic studies over the past decade have resulted in a putative mechanism of action for the GroEL chaperonin [19]. Central to this mechan...

The MitCHAP-60 Disease Is Due to Entropic Destabilization of the Human Mitochondrial Hsp60 Oligomer

“…3B). These results are in accord with previously published results (14). Notably, the oligomers of the D3G mutant were much more sensitive to dilution than the wild-type oligomers.…”

“…For this purpose, wild-type mHsp60 and the D3G mutant were expressed in bacteria and purified. In the last step of purification, we used gel filtration to separate active oligomeric forms of the protein from unassembled potentially inactive monomers (14,28). All examined preparations eluted in two peaks corresponding to monomers and oligomers (supplemental Fig.…”

“…In the case of the wild-type protein, the addition of mHsp10 resulted in 30% inhibition of its hydrolysis rate, whereas in the case of the D3G mutant, the ATPase activity was increased by 70% (although the co-chaperonin is expected to inhibit the ATPase activity by 50% (37)). Previous studies showed that mHsp10 stabilizes the oligomeric structure of the unstable mitochondrial and chloroplast chaperonins and, therefore, can cause an indirect activation of the ATPase activity as observed in the case of the D3G mutant (14,29). Interestingly, the rate of ATP hydrolysis by the N-del mutant was enhanced by 650% when mHsp10 was included.…”

“…The mitochondrial Hsp60 (mHsp60) 3 is similar to GroEL in that it is made up of heptameric rings (12-14) that can refold denatured substrates in vitro with the assistance of a co-chaperonin and ATP (15). However, the mHsp60 oligomer is less stable than the bacterial homolog, and it exhibits unique nucleotide binding properties and specificity for co-chaperonin (13,14,16).In addition to their essential function in mediating protein folding, the mammalian mitochondrial chaperonins were also suggested to be involved in extramitochondrial activities. A number of reports have suggested that mHsp60 can stimulate human leukocytes and vascular endothelial cells to produce proinflammatory cytokines (17).…”

“…The mitochondrial Hsp60 (mHsp60) 3 is similar to GroEL in that it is made up of heptameric rings (12)(13)(14) that can refold denatured substrates in vitro with the assistance of a co-chaperonin and ATP (15). However, the mHsp60 oligomer is less stable than the bacterial homolog, and it exhibits unique nucleotide binding properties and specificity for co-chaperonin (13,14,16).…”