Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10 (original) (raw)
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Hsp60-independent protein folding in the matrix of yeast mitochondria
The EMBO journal, 1996
Proteins that are imported from the cytosol into mitochondria cross the mitochondrial membranes in an unfolded conformation and then fold in the matrix. Some of these proteins require the chaperonin hsp60 for folding. To test whether hsp60 is required for the folding of all imported matrix proteins, we monitored the folding of four monomeric proteins after import into mitochondria from wild-type yeast or from a mutant strain in which hsp60 had been inactivated. The four precursors included two authentic matrix proteins (rhodanese and the mitochondrial cyclophilin Cpr3p) and two artificial precursors (matrix-targeted variants of dihydrofolate reductase and barnase). Only rhodanese formed a tight complex with hsp60 and required hsp60 for folding. The three other proteins folded efficiently without, and showed no detectable binding to, hsp60. Thus, the mitochondrial chaperonin system is not essential for the folding of all matrix proteins. These data agree well with earlier in vitro st...
Identification of Elements That Dictate the Specificity of Mitochondrial Hsp60 for Its Co-Chaperonin
PLoS ONE, 2012
Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial cochaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all cochaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial cochaperonin.
The human mitochondrial Hsp60 in the APO conformation forms a stable tetradecameric complex
Cell Cycle, 2017
The human mitochondrial chaperonin is a macromolecular machine that catalyzes the proper folding of mitochondrial proteins and is of vital importance to all cells. This chaperonin is composed of 2 distinct proteins, Hsp60 and Hsp10, that assemble into large oligomeric complexes that mediate the folding of non-native polypeptides in an ATP dependent manner. Here, we report the bacterial expression and purification of fully assembled human Hsp60 and Hsp10 recombinant proteins and that Hsp60 forms a stable tetradecameric double-ring conformation in the absence of co-chaperonin and nucleotide. Evidence of the stable double-ring conformation is illustrated by the 15 A resolution electron microscopy reconstruction presented here. Furthermore, our biochemical analyses reveal that the presence of a nonnative substrate initiates ATP-hydrolysis within the Hsp60/10 chaperonin to commence protein folding. Collectively, these data provide insight into the architecture of the intermediates used by the human mitochondrial chaperonin along its protein folding pathway and lay a foundation for subsequent high resolution structural investigations into the conformational changes of the mitochondrial chaperonin.
Journal of Biological Chemistry, 2010
Mitochondria biogenesis requires the import of several precursor proteins that are synthesized in the cytosol. The mitochondrial heat shock protein 70 (mtHsp70) machinery components are highly conserved among eukaryotes, including humans. However, the functional properties of human mtHsp70 machinery components have not been characterized among all eukaryotic families. To study the functional interactions, we have reconstituted the components of the mtHsp70 chaperone machine (Hsp70/J-protein/GrpE/Hep) and systematically analyzed in vitro conditions for biochemical functions. We observed that the sequence-specific interaction of human mtHsp70 toward mitochondrial client proteins differs significantly from its yeast counterpart Ssc1. Interestingly, the helical lid of human mtHsp70 was found dispensable to the binding of P5 peptide as compared with the other Hsp70s. We observed that the two human mitochondrial matrix J-protein splice variants differentially regulate the mtHsp70 chaperone cycle. Strikingly, our results demonstrated that human Hsp70 escort protein (Hep) possesses a unique ability to stimulate the ATPase activity of mtHsp70 as well as to prevent the aggregation of unfolded client proteins similar to J-proteins. We observed that Hep binds with the C terminus of mtHsp70 in a full-length context and this interaction is distinctly different from unfolded client-specific or J-protein binding. In addition, we found that the interaction of Hep at the C terminus of mtHsp70 is regulated by the helical lid region. However, the interaction of Hep at the ATPase domain of the human mtHsp70 is mutually exclusive with J-proteins, thus promoting a similar conformational change that leads to ATPase stimulation. Additionally, we highlight the biochemical defects of the mtHsp70 mutant (G489E) associated with a myelodysplastic syndrome.
A single-ring mitochondrial chaperonin (Hsp60-Hsp10) can substitute for GroEL-GroES in vivo
Journal of bacteriology, 1999
Chaperonins participate in the facilitated folding of a variety of proteins in vivo. To see whether the same spectrum of target proteins can be productively folded by the double-ring prokaryotic chaperonin GroEL-GroES and its single-ring human mitochondrial homolog, Hsp60-Hsp10, we expressed the latter in an Escherichia coli strain engineered so that the groE operon is under strict regulatory control. We found that expression of Hsp60-Hsp10 restores viability to cells that no longer express GroEL-GroES, formally demonstrating that Hsp60-Hsp10 can carry out all essential in vivo functions of GroEL-GroES.
Cryo-EM reveals the dynamic interplay between mitochondrial Hsp90 and SdhB folding intermediates
bioRxiv, 2020
TRAP1 is a mitochondrion specific Hsp90, a ubiquitous chaperone family that mediates the folding and maturation of hundreds of “client” proteins. Through the interaction with client proteins, TRAP1 regulates mitochondrial protein homeostasis, oxidative phosphorylation/glycolysis balance, and plays a critical role in mitochondrial dynamics and disease. However, the molecular mechanism of client protein recognition and remodeling by TRAP1 remains elusive. Here we established the succinate dehydrogenase B subunit (SdhB) from mitochondrial complex II as a client protein for TRAP1 amenable to detailed biochemical and structural investigation. SdhB accelerates the rate of TRAP1 dimer closure and ATP hydrolysis by 5-fold. Cryo-EM structures of the TRAP1:SdhB complex show TRAP1 stabilizes SdhB folding intermediates by trapping an SdhB segment in the TRAP1 lumen. Unexpectedly, client protein binding induces an asymmetric to symmetric transition in the TRAP1 closed state. Our results highligh...
Ecm10, a novel Hsp70 homolog in the mitochondrial matrix of the yeast Saccharomyces cerevisiae
Febs Letters, 2000
Members of the heat shock protein 70 (Hsp70) family are found in most of the compartments of eukaryotic cells where they play essential roles in protein metabolism. In yeast mitochondria, two Hsp70 proteins are known: Ssc1 and Ssq1. We identified Ecm10 as a third Hsp70 protein in the mitochondrial matrix. Ecm10 shares 82% amino acid identity with Ssc1 and 54% with Ssq1. Overexpression of Ecm10 mitigates protein import defects in ssc1 mutants suggesting that Ecm10 can play a role in protein translocation. Like Ssc1, Ecm10 interacts with the nucleotide exchange factor Mge1 in an ATP-dependent manner. Deletion of ecm10 leads to synthetic growth defects with ssc1 mutations at low temperature. Our data suggest an overlapping function of Ecm10 and Ssc1. ß
Identification of a groES-like chaperonin in mitochondria that facilitates protein folding
Proceedings of the National Academy of Sciences, 1990
Mitochondria contain a polypeptide that is functionally equivalent to Escherichia coli chaperonin 10 (cpn10; also known as groES). This mitochondrial cpn10 has been identified in beef and rat liver and is able to replace bacterial cpn10 in the chaperonin-dependent reconstitution of chemically denatured ribulose-1,5-bisphosphate carboxylase. Thus, like the bacterial homologue, mitochondrial cpn10 facilitates a K(+)- and Mg.ATP-dependent discharge of unfolded (or partially folded) ribulose bisphosphate carboxylase from bacterial chaperonin 60 (cpn60; also known as groEL). Instrumental to its identification, mitochondrial cpn10 and bacterial cpn60 form a stable complex in the presence of Mg.ATP. Bacterial and mitochondrial cpn10 compete for a common saturable site on bacterial cpn60. As a result of complex formation, with either mitochondrial or bacterial cpn10, the "uncoupled ATPase" activity of bacterial cpn60 is virtually abolished. The most likely candidate for mitochondr...